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WORLD INTELLECTUAL PROPERTY ORGANIZATION

International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 5 :

C07H 21/02, 21/04, C12N 15/70
C12N 15/74, 15/79, 5/10
C07K 15/28, A61K 39/395

(11) International Publication Number:
(43) International Publication Date:

WO 94/05690

17 March 1994(17.03.94)

(21) International Application Number: PCT/US93/08435

(22) International Filing Date: 8 September 1993 (08.09.93)

(30) Priority data:

07/941,654

9 September 1992 (09.09.92) US

(60) Parent Application or Grant

(63) Related by Continuation
US

Filed on

07/941,654 (CIP)
9 September 1992 (09.09.92)

(71) Applicants (for all designated States except US): SMITH-
KLINE BEECHAM CORPORATION [US/US]; 709
Swedeland Road, King of Prussia, PA 19406-2799 (US).
UNITED STATES OF AMERICA as represented by
THE SECRETARY OF THE NAVY [US/US]; Pentag-
on, Washington, DC 20031 (US). UNITED STATES OF
AMERICA as represented by THE SECRETARY OF
THE ARMY [US/US]; Pentagon, Washington, DC
20031 (US).

(72) Inventors ; and

(75) Inventors/Applicants (for US only) : GROSS, Mitchell Stu-
art [US/US]; 667 Pugh Road, Wayne, PA 19087 (US).
ROSENBERG, Martin [US/US]; 241 Mingo Road,
Royersford, PA 19468 (US). SADOFF, Jerald, Charles
[US/US]; 1622 Kalmia Road, Washington, DC 20012
(US). HOFFMAN, Stephen [US/US]; 308 Argosy
Drive, Gaithersburg, MD 20878 (US). SYLVESTER,
Daniel, R. [US/US]; 42 Rossiter Avenue, Phoenixville,
PA 19460 (US). CHAROENVIT, Yupin [US/US]; 2833
Schoolhouse Circle, Silver Spring, MD 20902 (US).
HURLE, Mark, [US/US]; 641 Columbus Street, Bridge-
port, PA 19405 (US).

(74) Agents: BAK, Mary, E. et al.; Howson and Howson,
Spring House Corporate Center, P.O. Box 457, Spring
House, PA 19477 (US).

(81) Designated States: AU, CA, JP, KR, NZ, US, European
patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT,
LU, MC, NL, PT, SE).

Published

With international search report.

(54) Title: NOVEL ANTIBODIES FOR CONFERRING PASSIVE IMMUNITY AGAINST INFECTION BY A PATHOG-
EN IN HUMANS

(57) Abstract

Proteins and peptides derived from a murine P. falciparum monoclonal antibody, including synthetic humanized variable
light chain and variable heavy chain sequences, CDR peptides, and humanized antibodies useful in therapeutic methods and
compositions for conferring passive immunity to infection by a malaria-causing parasite are provided.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

Austria

Australia

Barbados

Belgium

Burkina Faso

Bulgaria

Benin

Brazil

Belarus

Canada

Central African Republic

Congo

Switzerland

Cote d'l voire

Cameroon

China

Czechoslovakia

Czech Republic

Germany

Denmark

Spain

Finland

France

Gabon

United Kingdom

Guinea

Greece

Hungary

Ireland

Italy

Japan

Democratic People's Republic

of Korea

Republic of Korea

Kazakhstan

Liechtenstein

Sri Lanka

Luxembourg

Latvia

Monaco

Madagascar

Mali

Mongolia

Mauritania

Malawi

Niger

Netherlands

Norway

New Zealand

Poland

Portugal

Romania

Russian Federation

Sudan

Sweden

Slovenia

Slovak Republic

Senegal

Chad

Togo

Ukraine

United States of America

Uzbekistan

Viet Nam

WO 94/05690

PCI7US93/08435

NOVEL ANTIBODIES FOR CONFERRING PASSIVE IMMUNITY
AGAINST INFECTION BY A PATHOGEN IN HUMANS

Field of the Invention

This invention relates generally to the field
5 of monoclonal and recombinant antibodies directed to

epitopes on selected pathogens, e.g., a malaria parasite,
methods for preparing and using, and compositions
employing, these antibodies.

Background of the Invention

10 Malaria is a severe and widespread disease,

caused by various species of the protozoan parasite genus
Plasmodium, including four species that infect man, e.g.,
P. falciparum, P. vivax, P. ovale and P. malariae [See,
e.g., V. Enea et al. , Science , 225 : 628-630 (1984)].

15 Malaria remains one of the most widespread and fatal
diseases in the world today because of the lack of an
effective vaccine and programs to control vector
populations, as well as new drug-resistant strains.
Generally, treatment of malaria relies heavily on

2 0 prophylactic drugs, such as the 4-aminoquinolines.

However, for most cases, drug resistance by P. falciparum
and the production of some undesirable side effects, have
undermined the efficacy of these drug therapies.

The focus of much research effort in the field
25 of malaria prophylaxis is the sporozoite form of the

Plasmodium parasite, particularly the circumsporozoite
(CS) protein [Clyde et al. , Am. J. Trop. Med. Hyq. .
24:397 (1975); Rieckman et al. , Bull. WHO . 57(1): 261
(1979); and U. S. Patent 4,957,869]. The cloning and

3 0 characterization of the CS protein genes or fragments

thereof of a number of Plasmodium species and recombinant
expression thereof in E. coli or yeast host cells have
been reported. The central repeat domain of the CS
proteins is immunodominant, i.e., if one injects

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sporozoites into an animal, the animal produces anti-
repeat antibodies. The first anti-sporozoite candidate
vaccine tested in man was based upon the repetitive
epitopes found on the CS protein of P. falciparum
5 consisting of (AsnAlaAsnPro) 37 (AsnValAspPro) 4 [SEQ ID NO:
1], which is invariant in a number of strains examined to
date. Clinical trials utilizing a vaccine candidate,
called R32tet32, consisting of NH 2 -Met-Asp-Pro-[ (Asn-Ala-
Asn-Pro) 15 (Asn-Val -Asp-Pro) 1 ] 2 -Leu-Arg-Arg-Thr-His-Arg-

10 Gly-Arg-His-His-Arg-Arg-His-Arg-Cys-Gly-Cys-Trp-Arg-Leu-
Tyr-Arg-Arg-His-His-Arg-Trp-Gly-Arg-Ser-Gly-Ser-COOH [ SEQ
ID NO: 2], produced a protective response in a human
volunteer against the sporozoite challenge [see, Ballou
et al. , The Lancet , June 6, 1987, pp. 1277-1281; and

15 European Patent Publication No. 0192626, published August
27, 1986, incorporated herein by reference].

Numerous monoclonal antibodies (mAbs) directed
toward proteins from various stages of the Plasmodium
life cycle have been identified and shown to be effective

2 0 in passive transfer experiments in mice and monkeys [Y.

Charoenvit et al. , J. Immunol. . 146 (3) : 1020-1025 (1990)].
However, the use of antibodies for the treatment or
prophylaxis of malaria may have disadvantages. The
administration of murine or other animal antibodies to

25 humans may be limited by the adverse immune response of

humans to the foreign antibody, e.g., rapid clearance and
toxic side effects. Such immune responses in humans have
been shown to be directed against both immunoglobulin
constant and variable regions of murine antibodies.

30 Several techniques have been described which

suggest alteration of murine (and other species)
antibodies to reduce the occurrence of an immune response
in a desired species, e.g., human, to the parent antibody
[See, e.g., PCT Patent Publication No. PCT/WO86/0153 3 ,

35 published March 13, 1986; British Patent Application No.

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GB2188638A, published October 7, 1987; Amit et al. ,
Science , 233:747-753 (1986); Queen et al. , Proc. Natl.
Acad. Sci. USA, 86:10029-10033 (1989); PCT Patent
Publication No. PCT/WO90/07861, published July 26, 1990;
5 and Riechmann et al . , Nature . 332:323-327 (1988)]. While
the prior art suggests possible experimental techniques,
none show how to provide the combination of properties
required for effective prevention of in vivo growth of P.
fal ci parum .

10 There remains a need in the art for alternative

methods of providing immunity against infection with
selected pathogens, e.g., a malarial parasite,
particularly for a prophylactic agent capable of
providing effective short-term protection.

15 Summary of the Invention

In one aspect, the present invention
provides complementarity determining region (CDR)
peptides from a monoclonal antibody directed against a
selected epitope on a pathogen, as well as fragments and

20 analogs of these peptides. Preferably, the antibody is

capable of binding an epitope of Plasmodium, particularly
the CS repeat region epitope or a fragment thereof, e.g.,
murine anti-P. falciparum mAb NFS2. These CDRs retain
the antigen binding specificity of the mAb from which
25 they were derived.

Another aspect provides an isolated, naturally
occurring or synthetic, humanized immunoglobulin light or
heavy chain variable region amino acid sequence
comprising one or more CDR sequences originating from the
30 light or heavy chain of such a selected antibody.

In yet a further aspect, the invention provides
a fusion protein comprising a first amino acid sequence
derived from the variable light chain and/or heavy chain
of an ant i -Plasmodium antibody, an ant i -Plasmodium CDR, a

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functional fragment or analog thereof. The first
selected amino acid sequence is operatively linked or
fused to a second selected amino acid sequence. These
fusion proteins are characterized by the antigen binding
5 specificity of the mAb from which the first selected
amino acid sequence is derived.

A further aspect of the invention provides an
engineered antibody with specificity for the selected
Plasmodium epitope, e.g., P. falciparum repeat region.

10 In another aspect, the invention provides a P.

falciparum antibody or fragment thereof produced by
screening hybridoma products derived from any species
immunoglobulin repertoires, or human or murine antibody
combinatorial libraries, with the epitope of mAb NFS2.

15 In a further aspect, the present invention

provides F ab fragments of the above-described engineered
antibodies or ant i -Plasmodi urn mAbs.

As yet additional aspects, the invention
provides nucleic acid sequences which encode the

20 proteins, peptides, antibodies and fragments described

herein, as well as plasmids containing one or more of the
sequences, host cells transformed therewith, and methods
for producing the products of expression of these
nucleotide sequences in host cells, e.g., mammalian

25 cells.

Other aspects provided by the invention include
a pharmaceutical composition and a prophylactic method
for conferring passive immunity to a human anticipating
exposure to a malarial parasite, comprising an effective

30 amount of at least one protein, antibody, peptide or
fragment described herein and a pharmaceutically
acceptable carrier or diluent.

Other aspects and advantages of the present
invention are described further in the following detailed

3 5 description of preferred embodiments thereof.

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Brief Des cription of the Drawings

Fig. 1 illustrates the amino acid SEQ ID NO: 4
and nucleotide SEQ ID NO: 3 sequences of the naturally
occurring light chain variable region of mAb NFS2.

Fig. 2 illustrates the amino acid SEQ ID NO: 6
and nucleotide SEQ ID NO: 5 sequences of a synthetic
humanized light chain variable region Pfhzlcl-l
containing anti-PIasmodiura CDRs SEQ ID NOs: 21-26. The
CDRs are underlined.

Fig. 3 illustrates the amino acid SEQ ID NO: 8
and nucleotide SEQ ID NO: 7 sequences of synthetic
humanized light chain variable region Pfhzlcl-2.

Fig. 4 illustrates the amino acid SEQ ID NO: 10
and nucleotide SEQ ID NO: 9 sequences of the naturally
15 occurring heavy chain variable region of mAb NFS2.

Fig. 5 illustrates the amino acid SEQ ID NO: 12
and nucleotide SEQ ID NO: 11 sequences of a synthetic
humanized heavy chain variable region Pfhzhc2-4.

Fig. 6 illustrates the amino acid SEQ ID NO: 14
20 and nucleotide SEQ ID NO: 13 sequences of synthetic
humanized heavy chain variable region Pfhzhc2-3.

Fig. 7 is a schematic drawing of plasmid
Pfhzhc2-3-Pcd employed to express a synthetic anti-
Plasmodium heavy chain in mammalian cells. The plasmid
25 contains a beta lactamase (Beta- lac) gene, an SV40 origin
of replication (SV40) , a cytomegalovirus promoter
sequence (CMV) , the synthetic heavy chain Pfhzhc2-3 SEQ
ID NO: 13, a poly A signal from bovine growth hormone
(BGH) , a betaglobin promoter (beta glopro) , a
30 dihydrofolate reductase gene (DHFR) , and another BGH
sequence poly A signal in a pUC19 background.

Fig. 8 is a schematic drawing of plasmid
Pfhzlcl-l-Pcn employed to express a synthetic light chain
in mammalian cells. The plasmid differs from that of
35 Fig. 7, in that it contains the synthetic humanized light

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chain Pfhzlcl-l SEQ ID NO: 5 rather than the heavy chain,
and a neomycin gene (Neo) in place of DHFR.

Fig. 9 illustrates the nucleotide SEQ ID NO: 42
and amino acid SEQ ID NO: 43 sequences of a synthetic
5 humanized heavy chain variable region Pfhzhc2-6.

Detailed Descript ion of the Invent-i nn

The present invention provides prophylactic
agents capable of conferring a short duration, protective
10 immune state against infection of humans by selected
pathogens in the immunized human, e.g., for epidemic
control and for use by those anticipating exposure to the
pathogen. Recombinant or engineered antibodies,
preferably chimeric, humanized or human monoclonal
15 antibodies, are capable of use as such passive protective
proteins. These proteins in a prophylactic composition
may be administered before anticipated exposure to the
pathogen and would not require daily regimens of follow-
up doses to mediate the short term protection.
20 While the following description refers

specifically to antibodies capable of conferring passive
protection to the sporozoite form of the pathogen, P.
falciparum, a causative agent of malaria in humans, the
invention described herein is not limited to any
25 particular stage of that pathogen nor to that pathogen

alone. The teachings of the present invention permit one
skilled in the art to construct other recombinant
antibodies directed to other selected pathogens, e.g.,
other species of Plasmodium, including the blood stages,
liver stages, or gametocyte stages. Antibodies of the
invention directed against the circumsporozoite CS gene
of the other human infective parasites , e.g., p.
malar iae, P. vivax and P. ovale, may also be constructed
according to this invention to provide passive transfer
35 proteins useful against these parasitic infections.

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Similarly, passive therapy agents prepared according to
the invention may involve other infective agents,
viruses, bacteria and the like. Additionally, such
antibodies may also be useful as therapeutic agents for
5 the treatment of acute stages of infections.

I. Definitions

"First fusion partner" refers to a nucleic acid
seguence encoding an amino acid seguence, which can be
all or part of an immunoglobulin heavy chain, a light
10 chain, functional fragment thereof including the variable
region from one or both chains and CDRs therefor, or an
analog thereof, having the antigen binding specificity of
a selected high titer antibody, preferably the murine
antibody, NFS2 .

15 "Second fusion partner" refers to another

nucleotide seguence encoding a protein or peptide to
which the first fusion partner is fused in frame or by
means of an optional conventional linker seguence. Such
second fusion partner is preferably heterologous to the
20 first fusion partner. A second fusion partner may
include a nucleic acid seguence encoding a second
antibody region of interest, e.g., all or part of an
appropriate human constant region or framework region.

"Fusion molecule" refers to the product of a
25 first fusion partner operatively linked to a second
fusion partner. "Operative linkage" of the fusion
partners is defined as an association which permits
expression of the antigen specificity of the anti-P.
falciparum seguence (the first fusion partner) from the
30 donor antibody as well as the desired characteristics of
the second fusion partner. For example, a nucleic acid
seguence encoding an amino acid linker may be optionally
used, or linkage may be via fusion in frame to the second
fusion partner.

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"Fusion protein" refers to the protein encoded
by the fusion molecule, which may be obtained by
expression of the fusion molecule in a selected host
cell. Such fusion proteins may be engineered antibodies,
5 e.g., chimeric or humanized antibodies, or any of the
antibody regions identified herein fused to
immunoglobulin or non-immunoglobulin proteins and the
like.

"Donor antibody" refers to an antibody
10 (polyclonal, monoclonal or recombinant) which contributes
its naturally-occurring or modified variable light and/or
heavy chains, variable regions thereof, CDRs thereof or
other functional fragments thereof to a first fusion
partner, so as to provide the fusion molecule and fusion
15 protein, with the antigenic specificity characteristic of
the donor antibody. One donor antibody suitable for use
in this invention is murine mAb NFS2 and others are
described below.

"Acceptor antibody" refers to an antibody

20 (polyclonal, monoclonal or recombinant) heterologous to
the donor antibody, but homologous to the patient (human
or other mammal) to be treated, which contributes all or
any portion of the sequences of its variable heavy and/ or
light chain framework regions and/ or its heavy and/ or

25 light chain constant regions to a second fusion partner.
Preferably a human antibody is the acceptor antibody.

"CDRs" are defined as the complementarity
determining region amino acid sequences of an antibody
which are the hypervariable regions of the heavy and

3 0 light chains. CDRs provide the majority of contact

residues for the binding of the antibody to the antigen
or epitope. CDRs of interest in this invention are
derived from donor antibody variable heavy and light
chain sequences, and include functional fragments and

35 analogs of the naturally occurring CDRs, which share or

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retain the same antigen binding specificity as the donor
antibody from which they were derived.

By "sharing the antigen binding specificity" it
is meant, for example, that although mAb NFS2 may be
5 characterized by a certain level of antigen affinity, and
a CDR encoded by a nucleic acid sequence of NFS2 in an
appropriate structural environment may have a lower
affinity, it is expected that CDRs of NFS2 in such
environments will nevertheless recognize the same
10 epitope (s) as NFS2.

A "functional fragment" is a partial CDR
sequence or partial heavy or light chain variable
sequence which retains the same antigen binding
specificity as the antibody from which the fragment was
15 derived.

An "analog" is an amino acid or peptide
sequence modified by replacement of at least one amino
acid, modification or chemical substitution of an amino
acid, which modification permits the amino acid sequence
20 to retain the biological characteristics, e.g., antigen
specificity, of the unmodified sequence.

An "allelic variation or modification" is an
alteration in the nucleic acid sequence encoding the
amino acid or peptide sequences of the invention. Such
25 variations or modifications may be due to degeneracies in
the genetic code or may be deliberately engineered to
provide desired characteristics. These variations or
modifications may or may not result in alterations in any
encoded amino acid sequence.

An "engineered antibody" is a type of fusion
protein, i.e., a synthetic antibody (e.g., a chimeric or
humanized antibody) in which a portion of the light
and/or heavy chain variable domains of a selected
acceptor antibody is replaced by analogous parts of CDRs
3 5 from one or more donor antibodies which have specificity

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for the selected epitope. These engineered antibodies
may also be characterized by alteration of the nucleic
acid sequences encoding the acceptor antibody light
and/or heavy variable domain framework regions in order
5 to retain donor antibody binding specificity. These

antibodies can comprise immunoglobulin constant regions
and variable framework regions from the acceptor
antibody, and one or more CDRs from the Plasmodium donor
antibodies described herein. Preferably the engineered
10 antibodies of the invention will be produced by
recombinant DNA technology.

"Chimeric antibody" refers to a type of
engineered antibody which contains naturally-occurring
variable region light chain and heavy chains (both CDR
15 and framework regions) derived from a non-human donor mAb
in association with light and heavy chain constant
regions derived from a human (or other heterologous
animal) acceptor mAb.

"Humanized antibody" refers to an engineered
20 antibody having its CDRs and/ or other portions of its
light and/ or heavy chain variable domain framework
regions derived from a non-human donor immunoglobulin,
the remaining immunoglobul in-derived parts of the
molecule being derived from one or more human
25 immunoglobulins. Such antibodies can also include

engineered antibodies characterized by a humanized heavy
chain associated with a donor or acceptor unmodified
light chain or a chimeric light chain, or vice versa.

"Effector agents" refers to non-protein carrier
3 0 molecules to which the fusion proteins, and/ or natural or
synthetic light or heavy chain of the donor antibody or
other fragments of the donor antibody may be associated
by conventional means. Such non-protein carriers can
include conventional carriers used in the diagnostic
35 field, e.g., polystyrene or other plastic beads, or other

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non-protein substances useful in the medical field and
safe for administration to humans and animals. Other
effector agents may include a macrocycle, for chelating a
heavy metal atom, or a toxin, such as ricin. Such
5 effector agents are useful to increase the half-life of
the anti-Plasraodium derived amino acid sequences or to
add to its properties,

II. Anti-Plasmodium Antibodies

For use in constructing the recombinant

10 antibodies of this invention as it relates to malaria-
causing pathogens, non-human species may be employed to
generate a desirable donor antibody upon presentment with
an antigen from a Plasmodium strain capable of infecting
humans. Conventional hybridoma techniques are employed

15 to provide a hybridoma cell line secreting a non-human

mAb to the selected antigen. As one example, the murine
mAb, NFS2, has been identified as a desirable antibody
which may be employed for use in developing a chimeric or
humanized antibody of this invention.

2 0 Murine IgG mAb NFS2 is characterized by an

antigen binding specificity to the repeat region of the
P. falciparum CS protein. In in vitro assays, it
prevented invasion of sporozoites into human hepatocytes
or hepatoma cells. Analogous antibodies in the mouse

25 model have conferred passive protection against malaria.
The production of mAb NFS2 is described in detail in
Example 1 below.

This invention is not limited to the use of the
illustrative NFS2 mAb or its hypervariable sequences.

30 Wherever in the following description the donor mAb is
identified as NFS2, this designation is made for
simplicity of description only. Other ant i -Plasmodium
antibodies may be substituted therefor. Suitable
antibodies include, for example, the murine mAb 2A10,

3 5 which is directed against the CS repeat protein or other

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mAbs described in R. A. Wirtz et al, Bull WHO . 65:39-45
(1987) .

Antibodies produced in other animals protected
by immunization with sporozoites or a protective epitope
5 of a selected Plasmodium species may be similarly

employed in this invention as a source of protective
ant i -PI asmodium sequences.

For example, the P. falciparum CS protein
repeat region protein R32tet32 NH 2 -Met-Asp-Pro- [ (Asn-Ala-

10 Asn-Pro) 15 (Asn-Val-Asp-Pro) x ] 2 -Leu-Arg-Arg-Thr-His-Arg-

Gly-Arg-His-His-Arg-Arg-His-Arg-Cys-Gly-Cys-Trp-Arg-Leu-
Tyr-Arg-Arg-His-His-Arg-Trp-Gly-Arg-Ser-Gly-Ser-COOH SEQ
ID NO: 2 may be employed to elicit both human and murine
mAbs with binding specificity therefor. This repeat

15 region protein is a suitable target for screening for
neutralizing antibodies useful in prophylactic agents
against malarial infection.

Similarly, the epitope to which NFS2 is
responsive, and analogs thereof, may be useful in the

2 0 screening and development of additional P. falciparum
antibodies, for use in the development of prophylactic
compositions for short-term protection of humans against
malaria. Other epitopes of interest include non-
repetitive flanking region epitopes, other repeat domains

25 or various liver, and blood and sexual stage epitopes of
Plasmodium species. Knowledge of these epitopes enables
one of skill in the art to define synthetic, and to
identify naturally-occurring, peptides which would be
suitable to confer passive or active immunity against P.

30 falciparum or other Plasmodium species. This knowledge
also permits the production of mAbs useful in the
prophylaxis of malarial infection in humans.

For example, other P. falciparum antibodies may
be developed by screening hybridomas or other

35 combinatorial libraries, or antibody phage displays [W.

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D. Huse et al ,, Science . 246:1275-1281 (1988)] using the
murine mAb epitope described herein. A collection of
antibodies, including hybridoma products or antibodies
derived from any species immunoglobulin repertoire may be
5 screened in a conventional competition assay, such as
described in the examples below, with one or more
epitopes described herein.

Antibodies such as those described above,
including those generated against a desired epitope and

10 produced by conventional techniques, including without
limitation, genes encoding murine mAbs, human mAbs, and
combinatorial antibodies, may be useful as donor
antibodies, as sources of antibody fragments, as well as
in prophylactic compositions against P. falciparum in

15 humans. Preferably, the antibodies developed in response
to Plasmodium, particularly P. falciparum, epitopes may
be useful as donors of desirable variable heavy and/or
light chain amino acid sequences, or functional fragments
thereof (e.g., CDRs) useful in the development of fusion

2 0 proteins, including engineered antibodies. Thus, the

invention may utilize a donor antibody, other than NFS2,
which is capable of binding to the P. falciparum peptide
consisting essentially of the amino acid sequence of the
repeat protein and analogs thereof.
25 Additionally, the mAbs identified herein, other

mAbs which are developed and are responsive to the use of
the sporozoites, R32tet32 [SEQ ID NO: 2] or the repeat
epitopes identified herein may be further altered or
manipulated to impart additional desirable prophylactic

3 0 characteristics .

III. Antibody Fragments, Amino Acid and Nucleotide
Sequences

The present invention provides isolated
naturally-occurring or synthetic variable light chain and
35 variable heavy chain sequences derived from mAb NFS2, as

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well as CDRs and fragments therefrom, which may be

employed in the design of fusion proteins (including

engineered antibodies) which are characterized by the

antigen binding specificity of this mAb.

5 The naturally-occurring variable heavy chain of

NFS2 is characterized by the amino acid and encoding

nucleic acid sequences illustrated in Fig. 4 [SEQ ID NOS:

9 and 10]. This chain is characterized by CDRs having

the following nucleotide and predicted amino acid

10 sequences. CDR 1 is characterized by the sequence:

AGCTATGCCATGTCT SEQ ID NO: 32
SerTyrAlaMetSer SEQ ID NO: 33.

The naturally occurring CDR 2 nucleic acid and amino acid

sequences are SEQ ID NOS: 17 and 18, respectively:

15 GAAATTAGTGATGGTGGTAGTTACACCTACTATCCAGACACTGTGACGGGC
Glul leSerAspGlyGlySerTyrThrTyrTyrProAspThrValThrGly .

The naturally-occurring CDR 3 has the nucleic acid and

amino acid sequences SEQ ID NOS: 19 and 20, respectively:

CTCATCTACTATGGTTACGACGGGTATGCTATGGACTAC
2 0 LeuIleTyrTyrGlyTyrAspGlyTyrAlaMetAspTyr .

Synthetic humanized variable heavy chains of

NFS2 are characterized by the amino acid and encoding

nucleic acid sequences illustrated in Fig. 5 [SEQ ID NOS:

9 and 10] and Fig. 6 [SEQ ID NOS: 13 and 14]. In both

25 synthetic chains, the CDRs have the following nucleotide

and predicted amino acid sequences. Nucleotide changes

were made in CDR 1 from the naturally occurring CDRs, and

are indicated by underlining. Synthetic CDR 1 is

characterized by the sequence:

30 AGCTATGCCATGAGC SEQ ID NO: 15
SerTyrAlaMetSer SEQ ID NO: 16.

The synthetic CDR 2 nucleic acid and amino acid sequences
are identical to the naturally-occurring sequences SEQ ID
NOS: 17 and 18 , respectively. The synthetic CDR 3 has
35 the same nucleic acid and amino acid sequences as does

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the naturally occurring CDR 3 SEQ ID NOS: 19 and 20,
respectively .

The naturally occurring variable light chain of
NFS2 is characterized by the amino acid sequence and
5 encoding nucleic acid sequence of Fig* 1 [SEQ ID NOS: 3
and 4]. This chain is further characterized by CDRs
having the following nucleotide and amino acid sequences.
CDR 1 is characterized by the nucleic acid and amino acid
sequences SEQ ID NOS: 34 and 35, respectively:
1 0 AAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAATTACTTGGCC

LysSerSerGlnSerLeuLeuTyrSerSerAsnGlnLysAsnTyrLeuAla.

CDR 2 is characterized by the nucleic acid and amino acid

sequences SEQ ID NOS: 36 and 37, respectively:

TGGGCATCCACTAGGGAATCT
15 TrpAlaSerThrArgGluSer •

CDR 3 is characterized by the nucleic acid and amino acid

sequences SEQ ID NOS: 38 and 39, respectively:

CAGCAATATTATAGCTATCCTCGGACG

GlnGlnTyrTyrSerTyrProArgThr .

20 A synthetic humanized variable light chain of

NFS2 is characterized by the amino acid and encoding
nucleic acid sequences illustrated in Fig. 2 [SEQ ID NOS:
5 and 6]. This chain is characterized by CDRs having the
following predicted amino acid sequences and encoded by

25 the illustrated nucleotide sequences. Nucleotide changes

were made in the three CDRs from the naturally occurring

corresponding CDRs, and are indicated by underlining.

Synthetic CDR 1 is characterized by the nucleic acid and

amino acid sequences SEQ ID NOS: 21 and 22, respectively:

3 0 AAGAGCT^TCAGAGCCTTTTATACTCGAGCAATCAAAAGAATTACTTGGCC
LysSerSerGlnSerLeuLeuTyrSerSerAsnGlnLysAsnTyrLeuAla.

Synthetic CDR 2 is characterized by the nucleic acid and

amino acid sequences SEQ ID NOS: 23 and 24, respectively:

TGGGCGTCAACTAGGGAATCT
3 5 TrpAlaSerThrArgGluSer .

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Synthetic CDR 3 is characterized by the amino

acid sequences SEQ ID NO: 26 and encoding nucleotide

sequences SEQ ID NO: 25, respectively:

CAGCAATATTATAGCTATCCGCGGACG
5 GlnGlnTyrTyrSerTyrProArgThr .

Another synthetic humanized variable light

chain of NFS2 is characterized by the amino acid and

encoding nucleic acid sequences illustrated in Fig. 3

[SEQ ID NOS: 7 and 8]. This chain is characterized by

10 identical CDRs 1 and 3 as in the Fig. 2 synthetic

sequences. However, a nucleotide change was made in the
CDR 2 from both the naturally occurring corresponding CDR
2, and the Fig. 2 synthetic CDR 2. Double underlining is
employed to indicate the change from the Fig. 2 synthetic

15 sequences. Synthetic CDR 2 is characterized by the

nucleic acid and amino acid sequences SEQ ID NOS: 40 and

4 1 , respectively : TGGGCGTCGACTAGGGAATCT

TrpAlaSerThrArgGluSer .

The present invention also includes the use of

2 0 F ab fragments or F (ab , )2 fragments. A F ab fragment

contains the entire light chain and amino terminal
portion of the heavy chain; and a F^ ab ,j 2 fragment is the
fragment formed by two F ab fragments bound by disulfide
bonds. MAb NFS2 and engineered antibodies derived
25 therefrom and described below provide sources of F ab

fragments and F (ab . )2 fragments which can be obtained by
conventional means, e.g. cleavage of the mAb with the
appropriate proteolytic enzymes, papain and/ or pepsin, or
by recombinant methods. The F ab fragments or F (ab , )2

3 0 fragments may be derived from any of the mAbs described

above, as agents protective in vivo against infection by
malarial pathogens, particularly P. falciparum.

The variable chain peptide sequences of murine
mAb NFS2, its variable chain peptide sequences and CDRs,
35 functional fragments, F ab fragments, and analogs thereof,
and the nucleic acid sequences encoding them, may be

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useful in obtaining various fusion molecules encoding
desired fusion proteins, particularly engineered
antibodies, and in methods for preparing and
administering pharmaceutical compositions containing
5 them •

The nucleic acid sequences of the invention, or
fragments thereof, encoding the variable light chains and
heavy chain peptide sequences or CDR peptides, or
functional fragments thereof are used in unmodified form

10 or are synthesized to introduce desirable modif ications.
The isolated naturally-occurring or synthetic nucleic
acid sequences, which are derived from mAB NFS2 or from
other desired anti-Plasmodium antibodies, may optionally
contain restriction sites to facilitate insertion or

15 ligation into a suitable nucleic acid sequence encoding a
desired antibody framework region, ligation with
mutagenized CDRs, or fusion with a nucleic acid sequence
encoding a selected second fusion partner.

Taking into account the degeneracy of the

20 genetic code, various coding sequences may be constructed
which encode the variable heavy and light chain amino
acid sequences, and CDR sequences of the invention, e.g.,
Figs. 1-6 [SEQ ID NOS: 3-26], and functional fragments
and analogs thereof which share the antigen specificity

25 of the donor antibody. The isolated or synthetic nucleic
acid sequences of this invention, or fragments thereof,
encoding the variable chain peptide sequences or CDRs or
functional fragments thereof can be used to produce
fusion proteins, i.e. chimeric or humanized antibodies,

3 0 or other engineered antibodies of this invention, when
operatively combined with a second fusion partner.

These sequences are also useful for mutagenic
insertion of specific changes within the nucleic acid
sequences encoding the CDRs or framework regions, and for

35 incorporation of the resulting modified or fusion nucleic

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acid sequence into a vector for expression. For example,
silent nucleotide substitutions may be made in the
nucleotide sequences encoding the CDRs to create
restriction enzyme sites to facilitate insertion of the
5 mutagenic frameworks, or to modify the selected

frameworks at nucleotide positions analogous to those of
the donor antibody. Such mutations may include those
inserted for the purpose of contributing to higher
antigen binding affinity.

10 IV. Fusion Molecules and Fusion Proteins

Fusion molecules of this invention can encode
engineered antibodies, chimeric antibodies and humanized
antibodies. A desired fusion molecule may contain a
first fusion partner encoding an amino acid sequence

15 having the antigen specificity of a Plasmodium antibody
directed against the amino acid sequence of the repeat
protein and analogs thereof, operatively linked to a
second fusion partner. Desirably the source of the first
fusion partner is a selected mAb, e.,g., mAb NFS2, the

2 0 source of nucleic acid sequences of Figs. 1 [SEQ ID NO:
3] and 4 [SEQ ID NO: 9].

A fusion molecule may encode an amino acid
sequence for a naturally occurring variable heavy chain
sequence of Fig. 4 [SEQ ID NOS: 9 and 10], a functional

25 fragment or analog thereof, a naturally occurring

variable light chain sequence of Fig. 1 [SEQ ID NO: 3 and
4], a functional fragment or analog thereof, or one or
more NFS2 CDRs [SEQ ID NO: 15-26]. Another exemplary
fusion molecule may encode a synthetic variable heavy

30 and/or light chain from the donor mAb, such as those of

Figs. 2 [SEQ ID NOS: 5 and 6], 3 [SEQ ID NOS: 7 and 8], 5
[SEQ ID NOS: 11 and 12], and 6 [SEQ ID NOS: 13 and 14],
having the antigen specificity of P. falciparum antibody.

A desirable fusion molecule of this invention

35 may be characterized by encoding an amino acid sequence

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containing at least one, and preferably all of the CDRs
[SEQ ID NOS: 15-2 6] of the variable region of the heavy
and/or light chains of the murine antibody NFS2, or a
functional fragment or analog thereof.
5 The second fusion partners are defined above,

and may include a sequence encoding a peptide, protein or
fragment thereof heterologous to the CDR-containing
sequence having the antigen specificity of NFS2 . One
example is a sequence encoding a second antibody region

10 of interest and may optionally include a linker sequence.

The resulting fusion molecule may encode both
anti-P. falciparum antigen specificity and the
characteristic of the second fusion partner, e.g., a
functional characteristic such as secretion from a

15 recombinant host, or a therapeutic characteristic if the
fusion partner itself encodes a therapeutic protein, or
additional antigenic characteristics, if the fusion
partner encodes a protein having its own antigen
specificity.

20 If the second fusion partner is derived from

another antibody, e.g., any isotype or class of
immunoglobulin framework or constant region (preferably
human) , or the like, an engineered antibody is provided.
Thus, for example, a fusion molecule of this invention

25 may comprise a complete antibody molecule, having full

length heavy and light chains (Figs. 4 [SEQ ID NOS: 9 and
10] and 1 [SEQ ID NOS: 3 and 4]). For example, the
invention includes isolated naturally-occurring or
synthetic nucleic acid sequences, which may encode

3 0 variable region sequences, CDR peptides, fragments
thereof derived from desired Plasmodium mAbs, any
fragment of an engineered antibody, such as the F ab or
F (ab f )2 fragment, a heavy chain dimer, or any minimal
recombinant fragment thereof such as an F v or a single-

35 chain antibody (SCA) or any other sequence encoding a

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protein with the same specificity as the selected mAb,
e.g., the Plasmodium mAb NFS2.

The first fusion partner may also be associated
with effector agents as defined above, to which the first
5 fusion partner may be operatively linked by conventional
means, e.g., attached to the NFS2 encoding nucleic acids
by a covalent bridging structure.

Fusion or linkage between the first fusion
partners and the selected second fusion partner may be by

10 way of any suitable means, e.g., by conventional covalent
or ionic bonds, protein fusions, or heterobifunctional
cross-linkers, e.g., carbodiimide, glutaraldehyde, and
the like. Where the first fusion partner is associated
with an effector agent, non-proteinaceous, conventional

15 chemical linking agents may be used to fuse or join the
anti-P. falciparum amino acid sequences to the effector
agent. Such techniques are known in the art and readily
described in conventional chemistry and biochemistry
texts .

20 Additionally, conventional inert linker

sequences which simply provide for a desired amount of
space between the fusion partners or between the first
fusion partner and the effector agent may also be
constructed into the fusion molecule. The design of such

25 linkers is well known to those of skill in the art.

Expression of such fusion molecules results in
fusion proteins of this invention. One particularly
desirable type of fusion protein includes the engineered
antibody in which, at a minimum, fragments of the

30 variable heavy and/ or light domains of an acceptor mAb
have been replaced by analogous parts of the variable
light and/or heavy chains from one or more donor
monoclonal antibodies, which include the Plasmodium mAbs
described herein, such as NFS2.

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One example of a particularly desirable
engineered antibody is a humanized antibody, in which
CDRs from a desired donor murine mAb are inserted into
the framework regions of a human antibody. A preferred
5 donor antibody is one directed against a Plasmodium

epitope, preferably one specific for the repeat region
epitope of P. falciparum. A particularly preferred donor
antibody has all or a portion of the variable domain
amino acid sequences of NFS2 . In these humanized

10 antibodies one, two or preferably three CDRs from the
Plasmodium antibody heavy chain and/or light chain
variable regions are inserted into the framework regions
of a selected human antibody, replacing the native CDRs
of that latter antibody.

15 Preferably, the variable domains in both human

heavy and light chains have been altered by CDR
replacement. This engineered humanized antibody thus
preferably has the structure of a natural human antibody
or a fragment thereof. Such humanized antibodies may or

2 0 may not also include minimal alteration of the acceptor
mAb light and/ or heavy variable domain framework region
in order to retain donor mAb binding specificity. The
humanized antibody possesses the combination of
properties required for effective prevention and

25 treatment of infectious P. falciparum disease in animals
or man.

The remainder of the engineered antibody may be
derived from any suitable acceptor human immunoglobulin.
A suitable human antibody may be one selected from a

30 conventional database, e.g., the Kabat database, Los

Alamos database, and Swiss Protein database, by homology
to the nucleotide and amino acid sequences of the donor
antibody. A human antibody characterized by a homology
to the framework regions of the donor antibody (on an

35 amino acid basis) may be suitable to provide a heavy

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chain constant region and/ or a heavy chain variable
framework region for the insertion of the donor CDRs. A
suitable acceptor antibody capable of donating light
chain constant or variable framework regions may be
5 selected in a similar manner. It should be noted that
the acceptor antibody heavy and light chains are not
required to originate from the same human antibody*

Desirably, the heterologous framework and
constant regions are selected from the human

10 immunoglobulin classes and isotypes, such as IgG

(subtypes 1 through 4) , IgM, IgA and IgE. However, the
acceptor antibody need not comprise only human
immunoglobulin protein sequences. For instance, a gene
may be constructed in which a DNA sequence encoding part

15 of a human immunoglobulin chain is fused to a DNA
sequence encoding the amino acid sequence of a
polypeptide effector or reporter molecule.

As one example, an engineered antibody may be
encoded by a synthetic nucleic acid sequence encoding

20 CDRs of the variable light chain region of NFS2 or a

functional fragment thereof in place of at least a part
of the nucleic acid sequence encoding the light chain
variable region of an acceptor mAb, and a nucleic acid
sequence encoding CDRs of the variable heavy chain region

25 of NFS2 or a functional fragment thereof in place of at
least a part of the nucleic acid sequence encoding the
heavy chain variable region of an acceptor mAb, such as a
human antibody. The resulting humanized antibody is
characterized by the antigen binding specificity of mAb

3 0 NFS2.

Alternatively, the engineered antibody (or the
other monoclonal antibodies) of the invention may have
attached to it an effector or reporter molecule.
Alternatively, the procedure of recombinant DNA
3 5 technology may be used to produce an engineered antibody

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of the invention in which the F c fragment or CH3 domain
of a complete antibody molecule has been replaced by an
enzyme or toxin molecule.

It will be understood by those skilled in the
5 art that such an engineered antibody may be further
modified by changes in variable domain amino acids
without necessarily affecting the specificity of the
donor antibody. It is anticipated that heavy and light
chain amino acids may be substituted by other amino acids
10 either in the variable domain frameworks or CDRs or both.
Such engineered antibodies can be effective in prevention
of productive malaria (e.g., by P. falciparum) infection
in humans.

Additionally, the invention provides fusion
15 proteins which are chimeric antibodies, as defined above.
Such antibodies differ from the humanized antibodies
described above by providing the entire donor antibody
heavy chain and light chain variable regions, including
framework regions, e.g., Figs. 1 [SEQ ID NOS: 3 and 4]

2 0 and 4 [SEQ ID NOS: 9 and 10], fused to acceptor constant

regions for both chains.

V. Production of Proteins and Antibodies

A fusion molecule, recombinant antibody or
fusion protein of this invention is desirably constructed
25 by recombinant DNA technology using genetic engineering
techniques. The same or similar techniques may also be
employed to generate other embodiments of this invention,
e.g., to construct the chimeric or humanized antibodies,
the synthetic light and heavy chains, the CDRs, and the

3 0 nucleic acid sequences encoding them, as above mentioned.

A specific embodiment of the compositions of
this invention is set out in Example 3 below using the
CDRs of murine NFS2 and one or more selected human
antibody light and heavy chain framework regions.
35 Briefly described, a hybridoma producing the murine

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antibody NFS2 is conventionally cloned, and the cDNA of
its heavy and light chain variable regions is obtained by
techniques known to one of skill in the art, e.g., the
techniques described in Sambrook et al. , Molecular
5 Cloning (A Laboratory Manual) . 2nd edition, Cold Spring
Harbor Laboratory (1989) . The variable regions of the
NFS2 are obtained using PCR primers, and the CDRs are
identified using a known computer database, e.g, Kabat,
for comparison to other antibodies.

10 Homologous framework regions of a heavy chain

variable region from a human antibody were identified
using the same databases, e.g., Kabat, and a human
antibody having homology to NFS2 was selected as the
acceptor antibody. The sequences of synthetic heavy

15 chain variable regions containing the NFS2 CDRs within

the human antibody frameworks were designed with optional
nucleotide replacements in the framework regions for
restriction sites. This designed sequence was
synthesized by overlapping oligonucleotides, amplified by

20 polymerase chain reaction (PCR) , and corrected for
errors .

A suitable light chain variable framework
region was designed in a similar manner, resulting in two
synthetic light chain variable sequences containing the

25 NFS2 CDRs. See, Figs. 2 [SEQ ID NOS: 5 and 6] and 3 [SEQ
ID NOS: 7 and 8]. As stated above, the source of the
light chain is not a limiting factor of this invention.

These synthetic variable light and/ or heavy
chain sequences and the CDRs of mAb NFS2, and their

3 0 encoding nucleic acid sequences, are employed in the
construction of fusion proteins and engineered
antibodies, preferably humanized antibodies, of this
invention, by the following process. By conventional
techniques, a DNA sequence is obtained which encodes the

35 donor antibody variable heavy or light chain regions,

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wherein at least the CDRs and those minimal portions of
the acceptor mAb light and/ or heavy variable domain
framework region required in order to retain donor mAb
binding specificity as well as the remaining
5 immunoglobul in-derived parts of the antibody chain
derived from a human immunoglobulin.

A conventional expression vector or recombinant
plasmid is produced by placing these sequences encoding
the fusion protein in operative association with

10 conventional regulatory control sequences capable of
controlling the replication and expression in, and/or
secretion from, a host cell. Such regulatory sequences
may be readily selected by one of skill in the art and
are not intended as a limitation of the present

15 invention. Regulatory sequence include promoter

sequences, e.g., CMV promoter, and signal sequences which
can be derived by one of skill in the art from
antibodies .

A first vector can contain a sequence encoding

20 a light chain-derived polypeptide. Similarly, a second
expression vector is produced having a similar DNA
sequence which encodes a complementary antibody light or
heavy chain. Preferably at least the CDRs (and those
minimal portions of the acceptor mAb light and/or heavy

25 variable domain framework region required in order to
retain donor mAb binding specificity) of the variable
domain are derived from a donor antibody and the
remaining immunoglobulin-derived parts of the antibody
chain are derived from a human immunoglobulin in these

30 vectors. Preferably this second expression vector is

identical to the first, with the exception of the coding
sequences and selectable markers, to ensure that each
polypeptide chain is functionally expressed.

In another alternative, a single vector of the

35 invention may be used, the vector including the sequence

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encoding both light chain and heavy chain-derived
polypeptides. The DNA in the coding sequences for the
light and heavy chains may comprise cDNA or genomic DNA
or both.

5 A selected host cell is co-transf ected by

conventional techniques with both the first and second
vectors (or the single vector) to create the transf ected
host cell of the invention comprising both the
recombinant or synthetic light and heavy chains. The

10 transfected cell is then cultured by conventional

techniques to produce the engineered antibody of the
invention. The humanized antibody which includes the
association of both the recombinant heavy chain and/ or
light chain is screened from culture by appropriate

15 assay, such as an ELISA assay followed by the Inhibition
of Sporozoite Invasion (ISI) assay described in the
examples below. Similar conventional techniques may be
employed to construct other fusion proteins of this
invention.

20 Thus, the invention also includes a recombinant

plasmid containing the coding sequence of the fusion
molecule or engineered antibody of the invention. Such a
vector is prepared by conventional techniques and
suitably comprises the above-described DNA sequences and

25 a suitable promoter operatively linked to the DNA

sequences which encode the engineered antibody. Such a
vector is transfected into a mammalian cell via
conventional techniques.

Suitable vectors for the cloning and subcloning

30 steps employed in the methods and construction of the

compositions of this invention may be selected by one of
skill in the art. For example, the conventional pUC
series of cloning vectors, may be used. One vector used
is pUC19, which is commercially available from supply

35 houses, such as Amersham (Buckinghamshire, United

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Kingdom) or Pharmacia (Uppsala, Sweden) . Additionally,
any vector which is capable of replicating readily, has
an abundance of cloning sites and marker genes, and is
easily manipulated may be used for cloning. Thus, the
5 selection of the cloning vector is not a limiting factor
in this invention.

Similarly, the vectors employed for expression
of the engineered antibodies according to this invention
may be selected by one of skill in the art from any

10 conventional vector. The vectors also contain selected
regulatory sequences which are in operative association
with the DNA coding sequences of the immunoglobulin
regions and capable of directing the replication and
expression of heterologous DNA sequences in selected host

15 cells, such as CMV promoters. These vectors contain the
above described DNA sequences which code for the
engineered antibody or other fusion protein.
Alternatively, the vectors may incorporate the selected
immunoglobulin sequences modified by the insertion of

2 0 desirable restriction sites for ready manipulation.

The expression vectors may also be
characterized by marker genes suitable for amplifying
expression of the heterologous DNA sequences, e.g., the
mammalian dihydrof olate reductase gene (DHFR) or neomycin
25 resistance gene (neo R ) . Other preferable vector

sequences include a poly A signal sequence, such as from
bovine growth hormone (BGH) , and the betaglobin promoter
sequence (betaglupro) . The expression vectors useful
herein may be synthesized by techniques well known to

3 0 those skilled in this art.

The components of such vectors, e.g. replicons,
selection genes, enhancers, promoters, and the like, may
be obtained from natural sources or synthesized by known
procedures for use in directing the expression of the
35 recombinant DNA in a selected host. Other appropriate

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expression vectors of which numerous types are known in
the art for mammalian, bacterial, insect, yeast, and
fungal expression may also be selected for this purpose.

Two exemplary expression vectors employed in
5 the following examples for expression of the synthetic

heavy and light chain sequences are the mammalian vectors
Pfhzhc2-3-Pcd and Pf hzlcl-l-Pcn (see Figs, 7 and 8).
However, this invention is not limited to the use of
these illustrative pUC19-based vectors.

10 The present invention also encompasses a cell

line transfected with a recombinant plasmid containing
the coding sequences of the engineered antibodies or
other fusion protein described by this invention. Host
cells useful for the cloning and other manipulations of

15 these cloning vectors are also conventional. However,

most desirably, cells from various strains of E. coli are
used for replication of the cloning vectors and other
steps in the construction of the mAbs of this invention.

Suitable host cells or cell lines for the

2 0 expression of the engineered antibody or other protein of

the invention of this invention are preferably a
eukaryotic cell, and most preferably a mammalian cell,
such as a CHO cell or a myeloid cell. Other primate
cells may be used as host cells and, most desirably,
25 human cells are used, thus enabling the protein to be
modified with human glycosylation patterns.
Alternatively, other eukaryotic cell lines may be
employed. The selection of suitable mammalian host cells
and methods for transformation, culture, amplification,

3 0 screening and product production and purification are

known in the art. See, e.g., Sambrook et al. , cited
above .

Bacterial cells may prove useful as host cells
suitable for the expression of the recombinant mAbs of
3 5 the present invention. However, due to the tendency of

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proteins expressed in bacterial cells to be in an
unfolded or improperly folded form or in a non-
glycosylated form, any recombinant mAb produced in a
bacterial cell would have to be screened for retention of
5 antigen binding ability. If the protein expressed by the
bacterial cell was produced in a properly folded form,
that bacterial cell would be a desirable host. For
example, various strains of E. coli used for expression
are well-known as host cells in the field of

10 biotechnology. Various strains of B. subtil is ,

Streptomyces , other bacilli and the like may also be
employed in this method.

Where desired, strains of yeast cells known to
those skilled in the art are also available as host

15 cells, as well as insect cells and viral expression

systems. See, e.g. Miller et al. , Genetic Engineering .
8:277-298, Plenum Press (1986) and references cited
therein.

The general methods by which the vectors of the

2 0 invention may be constructed, transfection methods

required to produce the host cells of the invention, and
culture methods necessary to produce the fusion protein,
and preferably an engineered antibody of the invention
from such host cell are all conventional techniques.
25 Likewise, once produced, the fusion proteins, preferably
the engineered antibodies of the invention, may be
purified from the cell culture contents according to
standard procedures of the art, including ammonium
sulfate precipitation, affinity columns, column

3 0 chromatography, gel electrophoresis and the like. Such

techniques are within the skill of the art and do not
limit this invention.

The engineered antibody is then examined for in
vitro activity by use of an assay appropriate for the
35 selected pathogen. Presently conventional ELISA assay

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formats are employed to assess qualitative and
quantitative binding of the engineered antibody to the
R32tet32 epitope [SEQ ID NO: 2]. The ISI assay described
in Example 6 may also be employed. A similar assay, the
5 inhibition of hepatocyte invasion assay (ILSDA) , may be
performed [S. Mellouk et al. , Bull. WHO , Suppl. 68:52-58
(1990)]. Additionally, assays currently being developed
in the SCID mouse model may also be used to verify
efficacy prior to subsequent human clinical studies
10 performed to evaluate the persistence of the engineered
antibody in the body despite the usual clearance
mechanisms.

The examples below demonstrate the method for
constructing the humanized antibodies derived from the
15 murine mAb NFS2 . Following the procedures described for
humanized antibodies prepared from this antibody, one of
skill in the art may also construct humanized antibodies
from other malarial antibodies, variable region sequences
and CDR peptides described herein. Engineered antibodies

2 0 can be produced with variable region frameworks

potentially recognized as "self" by recipients of the
altered antibody. Minor modifications to the variable
region frameworks can be implemented to effect large
increases in antigen binding without appreciable
25 increased immunogenicity for the recipient. Such

engineered antibodies can effectively passively protect a
human against P. falciparum infection.
VI. Therapeutic /Prophylactic Uses

The fusion proteins, particularly the

3 0 engineered antibodies described above, functional

fragments, analogs and the other protein or peptides
described herein may be employed as prophylactic agents,
capable of conferring short-term passive immunity to
infection by the pathogen from which the original
35 antigenic substance derives, e.g., P. falciparum, to a

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subject. The protective effect conferred by the use of
the engineered antibodies of this invention is produced
by binding of the immunoglobulin to the pathogen and the
subsequent removal of this bound complex by the normal
5 function of macrophages. Thus, the engineered antibodies
of the present invention, when in preparations and
formulations appropriate for prophylactic use, are highly
desirable for persons anticipating short-term exposure to
the pathogen, e.g., travelers , tourists, or military

10 personnel anticipating travel in endemic areas.

Therefore, this invention also relates to a
method of prophylactic treatment of human P. falciparum
infection in a human in need thereof which comprises
administering an effective, protective dose of antibodies

15 including one or more of the engineered antibodies or
other fusion proteins described herein, or fragments
thereof, to a human anticipating exposure to a species of
Plasmodium.

The fusion proteins , including the engineered

2 0 antibodies or fragments thereof of this invention, may
also be used in conjunction with other antibodies,
particularly human mAbs reactive with other epitopes
responsible for the disease against which the engineered
antibody of the invention is directed. Similarly mAbs

25 reactive with other epitopes responsible for the disease
in a selected animal against which the antibody of the
invention is directed may also be employed in veterinary
compositions. Any antibody that is capable of operating
without interfering with the Plasmodium antibody of this

30 invention, e.g., antibodies to other malaria stages or to
different epitopes, are useful in these compositions.

The prophylactic agents of this invention are
believed to be desirable to confer protection to exposure
to the pathogen for from about 4 days to about 8 weeks,

35 without requiring booster dosages of the agent. This

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definition of 1 short-term 1 relates to the relative
duration of the recombinant antibodies of the present
invention in the human circulation.

The mode of administration of the prophylactic
5 agent of the invention may be any suitable route which
delivers the agent to the host. The fusion proteins,
including the engineered antibodies, and fragments
thereof, and pharmaceutical compositions of the invention
are particularly useful for parenteral administration,

10 i.e., subcutaneously, intramuscularly or intravenously.
However, the agent is preferably administered by
intramuscular injection.

Prophylactic agents of the invention may be
prepared as pharmaceutical compositions containing an

15 effective amount of the engineered antibody of the
invention as an active ingredient in a nontoxic and
sterile pharmaceutical^ acceptable carrier. In the
prophylactic agent of the invention, an aqueous
suspension or solution containing the engineered

2 0 antibody, preferably buffered at physiological pH, in a

form ready for injection is preferred. The compositions
for parenteral administration will commonly comprise a
solution of the engineered antibody of the invention or a
cocktail thereof dissolved in an acceptable carrier,
25 preferably an aqueous carrier. A variety of aqueous

carriers may be employed, e.g., saline, glycine, and the
like. These solutions are sterile and generally free of
particulate matter. These solutions may be sterilized by
conventional, well known sterilization techniques. The

3 0 compositions may contain pharmaceutically acceptable

auxiliary substances as required to approximate
physiological conditions such as pH adjusting and
buffering agents, etc. The concentration of the antibody
of the invention in such pharmaceutical formulation can

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vary widely, i.e., from less than about 0.5%, usually at
or at least about 1% to as much as 15 or 2 0% by weight
and will be selected primarily based on fluid volumes,
viscosities, etc., according to the particular mode of
5 administration selected.

Thus, a pharmaceutical composition of the
invention for parenteral, e.g., intramuscular injection,
could be prepared to contain 1 mL sterile buffered water,
and between about 50 to about 100 mg of an engineered

10 antibody of the invention. Similarly, a pharmaceutical
composition of the invention for intravenous infusion
could be made up to contain 250 ml of sterile Ringer's
solution, and 150 mg of an engineered antibody of the
invention. Actual methods for preparing parenterally

15 administrable compositions are well known or will be

apparent to those skilled in the art and are described in
more detail in, for example, Remington 1 s Pharmaceutical
Science , 15th ed. , Mack Publishing Company, Easton,
Pennsylvania .

2 0 It is preferred that the prophylactic agent of

the invention, when in a pharmaceutical preparation, be
present in unit dose forms. The appropriate
therapeutically effective dose can be determined readily
by those of skill in the art. To effectively prevent P.

25 falciparum infection in a human or other animal, one dose
of approximately 1 mg/kg to approximately 2 0 mg/kg of a
protein or an antibody of this invention should be
administered parenterally, preferably intramuscularly
(i.m.) and possibly intravenously (i.v.). Such dose may

30 be repeated at appropriate intervals during exposure.

The antibodies, engineered antibodies or
fragments thereof described herein can be lyophilized for
storage and reconstituted in a suitable carrier prior to
use. This technique has been shown to be effective with

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conventional immunoglobulins and art-known lyophilization
and reconstitution techniques can be employed.

Single or multiple administrations of the
pharmaceutical compositions can be carried out with
5 dosage levels and pattern being selected by the treating
physician. In any event, the pharmaceutical composition
of the invention should provide a quantity of the
engineered antibodies of the invention 'sufficient to
effectively prevent infection.

10 The following examples illustrate the

construction of exemplary engineered antibodies and
expression thereof in suitable vectors and host cells,
and are not to be construed as limiting the scope of this
invention. All amino acids are identified by

15 conventional codes or by full name, unless otherwise

indicated. All restriction enzymes, plasmids, and other
reagents and materials were obtained from commercial
sources unless otherwise indicated. All general cloning,
ligation and other recombinant DNA methodology were as

20 performed in Sambrook et al. , cited previously, or the
first edition thereof.

Example 1 - Description of Production of NFS2

Murine IgG mAb NFS2 was made by repeated
injection of P. falciparum sporozoites into mice followed

25 by B cell fusion with a myeloma cell line. The murine
mAb NFS2 is characterized by an antigen binding
specificity to the repeat region of the P. falcipairum CS
protein. Specifically, the NFS2 mAb binds to the
epitope, Pro Asn Ala Asn Pro Asn SEQ ID NO: 27. It is

30 possible that the antibody also binds to a larger, or
overlapping epitope on the repeat region.

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This murine mAb, in in vitro assays, prevented
invasion of sporozoites into human hepatocytes and
hepatoma cells. Analogous antibodies in the mouse model
have conferred passive protection against malaria and
5 have been observed to be highly potent [see, e.g., R. A.
Wirtz et al . . Bull WHO , 65:39-45 (1987), incorporated
herein by reference] . This antibody is available from
the U. S. Naval Medical Research Institute.

Example 2 - Cloning and Sequencing of NFS 2
10 - Cytoplasmic RNA was prepared by the method of

Favaloro et al. , Meth. Enzymol. , 65:718-749 (1980) from
NFS2, and hybridoma cell lines. The following primers
were used in the synthesis of Ig heavy (V H ) and light
(V L ) chain variable region cDNAs, respectively. The V L
15 primers, #2580 and #2789, extended from Hindll through
Xbal and were made to conserved regions of murine RNA.

Hindlll

#2580: 5 » CCAGATGTAAGCTT CAGCTG ACCCAGTCTCCA3 1 SEQ ID NO: 28
2 0 PvuII

Xba I Nael

#2789:

5 ' CATCTAGATGGCGCCGCCACAGTACGTTTGATCTCCAGCTTGGTCCC3 1 SEQ
ID NO: 29 The V H primers, #2621 and #2853, extended from
25 Kpn l through PstI and were made to conserved regions of
murine RNA.

Kpnl Xhol
#2621: 5 1 GGGGTACC AGGTCC A ( A / G ) CT ( G / T ) CTCGAGTC ( A / T ) GG 3 "
SEQ ID NO: 30

30 PstI

#2853: 5 1 GCCTGC AGCTAGC TGAGGAGACGGTGACCGTGGTCCCTTGG-

Nhel

CCCCAG3 1 SEQ ID NO: 31
PCR, as described by Saiki et al. , Science ,
35 239 : 487-491 (1988), was performed on the RNA template.

For the PCR, the primers used were identified above. For

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PCR amplification of V H , DNA/primer mixtures consisted of
5 ill RNA and 0.5 /xM of the primers. For PCR
amplifications of V L/ DNA/primer mixtures consisted of 5
/il RNA and 0.5 MM of the primers. To these mixtures was
5 added 250 fM each of dATP, dCTP, dGTP and dTTP, 10 mM
Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl 2/ 0.01% (w/v)
gelatin, 0.01% (v/v) Tween 20, 0.01% (v/v) Nonidet P40
and 5 units AmpliTaq [Cetus] . Samples were subjected to
25 thermal cycles of PCR at 94°C, 30 seconds; 55°C, 30

10 seconds; 72°C, 45 seconds; ending with 5 minutes at 72°C.
For cloning and sequencing, amplified V H DNA was purified
on a low melting point agarose gel and by Elutip-d column
chromatography [Schleicher and Schuell-Dussel, Germany]
and cloned into pUC18 [New England Biolabs] . The general

15 cloning and ligation methodology was as described in
Maniatis et al., cited above.

V H DNA was cloned as Kpn I- Pst I fragments into
similarly-digested pUC18. V L DNA was cloned as Hindlll-
Xba l fragments into pUC18 digested with the same enzymes *

20 Representative clones were sequenced by the dideoxy
method [Sanger et al. , Proc. Natl. Acad. Sci. USA ,
Zi:5463-5467 (1977)] using T7 DNA polymerase [US
Biologicals] . From the sequences of NFS2 V H and V L
domains, the CDR sequences were elucidated in accordance

25 with the methodology of Kabat et al., in "Sequences of
Proteins of Immunological Interest", US Dept of Health
and Human Services, US Government Printing Office (1987)
utilizing computer assisted alignment with other V H and
V L sequences. The CDRs of the heavy and light chains of

30 NFS2 are listed above and identified herein as SEQ ID
NOS: 15-20 and 21-26.

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Example 3 - Humanized Antibodies

The following example describes the preparation
of an exemplary engineered antibody. Similar procedures
may be followed for the development of engineered
5 antibodies, using other Plasmodium antibodies or other

anti-pathogen antibodies developed by conventional means.

The source of the donor CDRs utilized to
prepare these engineered antibodies was the murine mAb,
NFS2, described in Examples 1 and 2 above. The sequenced

10 NFS2 variable framework regions were employed to again

search through the Kabat database to identify homologous
framework regions of a human antibody. The framework
region of an antibody obtained from a human SLE patient
B-cell hybridoma cell line 18/17 [H. Dersimonian et ah ,

15 J. Immunol . . 139 :2496-2501 (1987)] was determined to be
approximately 80% homologous to the NFS2 variable heavy
chain framework region.

Given the murine NFS2 CDRs (Example 2) and the
sequence of the human antibody 18/17, a synthetic heavy

20 chain variable region was made, and PCR performed to fill
in and amplify DNA. The NFS2 CDR sequences and the 18/17
V H framework regions were synthesized by the following
overlapping oligonucleotides:

SEQ ID NO: 44 : TAGTGAAGAATTCGAGGACGCCAGCAACATGGTGTTGCAGAC
25 CCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACGGGGAGGTGCAG
(Base 1-97) ;

SEQ ID NO : 4 5 : GCTAGCGGATTCACCTTTAGCAGCCATGTCGGACCCCCCAGG
GACTCTGAGAGGACACGTCGATCGCCTAAGTGGAAATCCTATGCCATGAGCTGGG
TCCGCCAGGCTCCAGGGAAAGGTCTAGAGTGGGTCTCAGAAATCTTTATAGTGAT

30 GGTGGTAGTTAC (Base 158-259);

SEQ ID NO: 46: GAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAG
GACACGTCTCTGTTAAGGTTCTTGTGCGACATAGACGTTTACTGCAGTATATTAC
TGTGCGAAACTCATCTACTATGGTTACGACGGGTATGCTATGGACTAGCTGCCCA
TACGATACCTGATC (Base 316-421) ;

35 SEQ ID NO: 47: TTCTTGAAAGCTTGGGCCCTTGGTACTAGCTGAGCTCACGG

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TGACCAGGGTACCCTGGCCCCAGTAGTCCATAGCATACCCGTCG ( Base 484-
400);

SEQ ID NO: 48: CATTTGCAGATACAGCGTGTTCTTGGAATTGTCTCTGGATA
TCGTGAACCGGCCCGTCACAGTGTCTGGATAGTAGGTGTAACTACCACCATCACT

5 AATTTC (Base 337-236) ;

SEQ ID NO: 49: CTAAAGGTGAATCCGCTAGCTGCACAGGAGAGTCTCAGGGA
CCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCGAGCAGCTGCACCTCCCCGTAG

GCACC (Base 177-77).

These primers are annealed together and DNA is

10 filled in using Taq polymerase, followed by PCR
amplification using the following 5 1 primer:
SEQ ID NO: 50: CCGCGAATTCGAGGACGCCAGCAAC
and 3 f primer: SEQ ID NO:51:
CCGCAAGCTTGGGCCCTTGGTACTAGCT .

15 Any errors in the mapped sequence which were

inserted by PCR were corrected. In addition,
conservative nucleotide replacements were placed in the
framework regions to introduce selected restriction sites
suitable for enzymatic cleavage. These alterations in

20 the framework regions are indicated by boxes in the

sequences of Figs. 2 [SEQ ID NOS: 5 and 6], 3 [SEQ ID
NOS: 7 and 8], 5 [SEQ ID NOS: 11 and 12] and 6 [SEQ ID
NOS: 13 and 14]. Additionally, most murine and human
antibodies have a basic residue before CDR3 . Because the

25 variable heavy chain of NFS2 has a non-basic residue Ser
before CDR3 [SEQ ID NOS: 19-20 and 25 and 26], the
acceptor basic residue (Lys) before CDR3 was deleted and
replaced with Ser to create heavy chain Pfhzhc2-3.

Two synthetic heavy chain variable regions were

30 obtained, namely, Pfhzhc2-3 SEQ ID NOS: 13 and 14, and
Pfhzhc2-4 SEQ ID NOS: 11 and 12. These sequences are
described in detail in Figs. 5 and 6. Each of these
synthetic heavy chain variable regions is characterized
by one or two nucleotide, or amino acid, differences 0

35 For example, Pfhzhc2-3 [SEQ ID NOS: 13 and 14] has a Ser

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at position 98; and Pfhzhc2-4 [SEQ ID NOS: 11 and 12] has
a Lys at position 98. Otherwise, these heavy chain
variable regions are identical.

For a suitable light chain variable framework
5 region, the NFS2 light chain CDRs and the light chain
variable framework sequence of the human antibody
identified in H. G. Klobeck et al. , Nucl. Acids Res. ,
13:6515-6529 (1985) were used to make a suitable
synthetic light chain sequence by the same methods. The
10 oligonucleotides used were as follows.

SEQ ID NO: 52: TAAGCGGAATTCGTAGTCGGATATCGTGATGACCCAGTC
TCCAGACTCGCTAGCTGTGTCTCTGGGCGAGAGGGC (Base 1-75) ;
SEQ ID NO: 53: TTACTTGGCCTGGTATCAGCAGAAACCCGGGCAGTCTCC
TAAGTTGCTCATAGTTTTCTTAATGAACCGGACTTACTGGGCGTCAACTAG (Base
15 130-198) ;

SEQ ID NO: 54: GACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAA
GATGTGGCAGTATACTACTGCTGTCTAAAGTGTCAGCAATATTATAGCTATCC

(Base 241-321) ;

SEQ ID NO: 55: CAGTTGGAAAGCTTGGCGCCGCCACAGTACGTTTGATCTCCA
20 CCTTGGTCCCTCCGCCGAACGTCCGCGGATAGCTATAATATTGC (Base 389-
304) ;

SEQ ID NO: 56: GTGAAATCTGTCCCAGACCCGCTGCCACTGAATCGG

TCAGGTACCCCAGATTCCCTAGTTGACGCC (Base 252-187) ;

SEQ ID NO: 57: CAGGCCAAGTAATTCTTTTGATTGCTCGAGTATAAA

25 AGGCTCTGAGAGCTCTTGCAGTTGATGGTGGCCCTCTCGCCC (Base 141-64) .

As described above, the primers were annealed
together and DNA filled in using Taq polymerase, followed
by PCR amplifcation with the following 5 1 SEQ ID NO: 58:
GCGGAATTCGTAGTCGGATATCGTGATGAC and 3 f SEQ ID NO: 59:

30 TGGAAAGCTTGGCGCCGCCACAGTACGTTTGATC primers.

Two synthetic light chain variable sequences
containing the NFS2 CDRs were designed and synthesized as
described above for the synthetic heavy chains and
referred to as Pfhzlcl-1 SEQ ID NOS: 5 and 6, and

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Pfhzlcl-2 SEQ ID NOS: 7 and 8. These two sequences
differed in amino acid sequence at only one amino acid
position, 49. Pfhzlcl-1 [SEQ ID NOS: 5 and 6] has a Ser
at position 49; Pfhzlcl-2 [SEQ ID NOS: 7 and 8] has a Pro
5 at the same position.

These synthetic variable light and/ or heavy
chain sequences are employed in the construction of an
exemplary humanized antibody. It is expected that any of
the synthetic heavy chains will successfully associate

10 with any of the synthetic light chains to produce a
useful humanized antibody.

To produce a humanized antibody, for the heavy
chain variable sequence Pfhzhc2-3 [SEQ ID NO: 13 and 14]
(Fig. 6) , the following signal sequence was synthesized

15 onto this variable region: SEQ ID NOS: 60: ATGGTGTTGCAG
ACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTAC, which
encodes SEQ ID NO: 61: MetValLeuGlnThrGlnValPhelleSerLeu
LeuLeuTrpIleSerGlyAlaTyr. For the synthetic light chain
variable sequence Pfhzlcl-1 [SEQ ID NO: 5 and 6] (Fig.

2 0 2) , the construct is digested with EcoRI and EcoRV, and

the same signal sequence was ligated onto the variable
sequence. Other signal sequences are well known to those
of skill in the art and may be substituted for this
exemplary sequence.
25 Selected constant regions of the human lgG 1

antibodies selected for the heavy and light chain were
synthesized and confirmed by PCR. These constant region
sequences were then inserted into pUC19-based expression
vectors. The above-described synthetic variable

3 0 constructs, containing the signal and variable regions of

the light and heavy chains, were thereafter inserted into
these pUC19-based expression vectors containing CMV
promoters and the constant regions and fused in frame to
the previously inserted human heavy and light chain

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constant regions by conventional methods [Maniatis et
ah . cited above]. Thus, after insertion of the
synthetic variable regions into these expression vectors,
the plasmids shown in Figs. 7 and 8, resulted. These
5 plasmids were then co-transf ected into a selected host

cell and, following incubation, the media was assayed for
antibody activity via ELISA as described in Example 4
below.

Using similar techniques, another exemplary
10 humanized antibody is constructed using the synthesized
heavy chain sequence Pfhzhc2-3 [SEQ ID NO: 13 and 14]
(Fig. 6) and the synthetic light chain sequence Pfhzlcl-2
[SEQ ID NO: 7 and 8] .

Example 4 - A High Affinity Humanized Antibody

15 The amino acid differences in the variable

regions of the frameworks of the original murine antibody
NSF2 described in Examples 1 and 2 and the Pf hzhc2 . 3 were
determined, and several changes were made to increase the
level of conservation of the original antibody

20 conformation.

At amino acid position 49, the Ser of the
humanized heavy chain Pfhzhc2.3 was changed to Ala, which
is the amino acid found at this position in the native
murine NSF2 . The replacement employed conventional

25 genetic engineering technology, e.g., by making a
synthetic DNA fragment containing the appropriate
nucleotide changes to alter the amino acid. A fragment
of Pf hzhc2 . 3 was digested with Xbal and EcoRV and the
synthetic fragment bearing the nucleotide change encoding

30 Ala in place of a Ser codon, is inserted to make the

amino acid replacement. The resulting synthetic heavy
chain was termed Pf hzhc2 . 6 .

This synthetic heavy chain was expressed as
previously described for the Pf hzhc2 . 3 synthetic heavy

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chain. The expression plasmid for this humanized heavy
chain sequence is essentially identical to the expression
plasmid illustrated in Fig. 7, with the exception of the
single amino acid difference described previously.
5 Similarly, humanized antibodies consisting of

the Pfhzhc2.6 heavy chain and Pfhzlcl.l light chain and
the Pfhzhc2.6 heavy chain and the Pfhzlcl.2 light chain
were assembled via co-transf ection of mammalian cells and
assayed for antibody activity by ELISA, as described in

10 Example 5 below.

Other high affinity antibodies specific for P.
falciparum can be developed using a similar method
designed to achieve minimal variable region framework
modifications. The method involves the following order

15 of steps of alteration and testing:

(1) In addition to the alteration at amino
acid position 49, other individual framework amino acid
residues known to be critical for interaction with CDRs
are compared in the primary antibody and the engineered

20 CDR-replacement antibody. For example, heavy chain amino
acid residue (Kabat numbering; see Kabat et al. , cited
above) is compared in the primary (donor) and engineered
antibodies. A residue at this position is thought to
interact with the invariant heavy chain CDR residue at

25 position 94 (Lys-basic) via a salt bridge.

If an amino acid is in the framework of the
donor antibody but not in the framework of the engineered
antibody, then an alternative heavy chain gene comprising
the engineered antibody is produced. In the reverse

30 situation whereby the engineered antibody framework
comprises a residue at one position but the donor
antibody does not, then an alternative heavy chain gene
comprising the original amino acid at that position is
reproduced. Prior to any further analysis, alternative

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plasmids produced on this basis are tested for production
of high affinity engineered antibodies.

(2) Framework amino acids within four residues
of the CDRs as defined according to Kabat (see Kabat et

5 al. , cited above) are compared in the primary antibody
and engineered CDR-replacement antibody. Where
differences are present, then for each region the
specific amino acids of that region are substituted for
those in the corresponding region of the engineered
10 antibody to provide a small number of engineered genes.
Alternative plasmids produced on this basis are then
tested for production of high affinity antibodies.

(3) Framework residues in the primary and
engineered CDR-replacement antibodies are compared and

15 residues with major differences in charge, size or

hydrophobicity are highlighted. Alternative plasmids are
produced on this basis with the individual highlighted
amino acids represented by the corresponding amino acids
of the primary antibody and such alternative plasmids are

2 0 tested for production of high affinity antibodies.

Example 5: ELISA Assays

Expression of the synthetic heavy chain and
light chain sequences were tested by transiently
transfecting the plasmid DNAs into monkey COS cells. The

25 following results are reported for Pfhzhc2-3 and Pfhzlcl-
1. Ten micrograms of the plasmids are mixed together and
ethanol precipitated. The DNAs are dissolved in Tris
buffered saline (TBS) and mixed with DEAE-dextran (400
/xg/ml final concentration) /chloroquine (0.1 mm), added to

30 3-4 x 10 5 COS cells grown in T25 flask, incubated for 4
hours at 37 °C. Cells were shocked with 10% DMSO in
phosphate buffered saline (PBS) for 1-2 minutes and after
washing with PBS, cells were incubated in the presence of
serum free growth medium. Media were collected 72 hours

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post transfection (day 3 sample) and fresh media were
added which were collected 120 hours post transfection
(referred to as day 5 sample) .

To compare the binding affinity of various
5 antibodies, i.e., chimeric and humanized, large scale COS
transf ections were performed as described above. For
each antibody, 200 fxg heavy chain plasmid and 2 00 /xg
light chain plasmid DNAs were used to transfect a total
of 2.5 x 10 7 COS cells. The media collected (day 3 and

10 day 5) were pooled, assayed for antibody expression using
F c capture ELISA. The media were concentrated using
Amicon to 6 ml. Amount of antibody in the pooled media
varied from 9 mg/ml to 25 mg/ml. These concentrated
samples were used to compare binding affinities via the

15 ISI and ILSDA assays.

The presence of humanized antibody in the
medium of wells containing transfected clones is measured
by conventional ELISA techniques. Micro-titer plates are
coated overnight at 4°C with goat anti-human IgG (F c

20 specific) antibodies [Sigma, St. Louis, MO] at 0.1 /xg per
well. After washing with PBS (pH 7.5), 50 /xl of culture
medium from the wells containing transf ectants is added
to each microtitre well for 2 hours at room temperature.
The wells are then emptied, washed with PBS and

25 peroxidase-conjugated goat anti-human IgG antibodies
[BioRad, Richmond, CA] are added at 50 jtiL of a 1/1000
dilution per well. Plates are then incubated at room
temperature for 1 hour. The wells are then emptied and
washed with PBS. 100 fxl 2.2 ' -azino-di[3-ethyl-

30 benzthiazoline sulfonate (6) ] are added per well.

Reactions were allowed to continue for 1 hour at room
temperature. The absorbance at 4 05 nm is then measured
spectrophotometrically . The ability of the humanized
antibody in the medium of wells containing transfected

35 clones to bind P. falciparum circumsporozoite protein was

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measured by ELISA. Microtiter plates were coated
overnight at 4°C with E. coli-produced R32tet32 at 0.1 fig
per well. After washing with PBS, 50 /xl of culture
medium from the wells which contain transf ectants is
5 added to each microtiter well for 2 hours at room

temperature. The wells are then emptied, washed with PBS
and peroxidase-conjugated goat anti-human IgG antibodies
[BioRad, Richmond, CA] are added at 50 ijlL of a 1/1000
dilution per well. Plates are then incubated at room

10 temperature for 1 hour. The wells are then emptied and
washed with PBS. 100 /il 2 . 2 1 -azino-di[3-ethyl-
benzthiazoline sulf onate(6) ] are added per well.
Reactions were allowed to continue for 1 hour at room
temperature. The absbrbance at 4 05 nm is then measured

15 spectrophotometrically .

In preliminary studies, an increase in affinity
was observed for the humanized antibodies of Example 4,
which contained the Pfhzhc2-6 heavy chain construct, as
compared to the Pfhzhc2-3 heavy chain construct.

2 0 Example 6 - Construction of Chimeric Antibody

A chimeric antibody of the invention was
constructed essentially as described above. A chimeric
antibody contains the native murine NSF2 variable
framework and CDR regions on the human constant regions
25 selected for the heavy chain [H. Dersimonian et al. , J.
Immunol . , 139 ; 2496-2501 (1987) and light chain [Klobeck
et ah , Nucl. Acids. Res. , 13:6515-6529 (1985)], with the
exception that the variable regions were obtained by PCR
of the RNA of the murine antibody obtained from the NFS2

3 0 hybridoma and the entire constant regions of the human

TqG 1 antibodies were synthesized by overlapping
oligonucleotides and amplified by PCR. Any errors which
were inserted by PCR were corrected. The resulting

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chimeric heavy chain and chimeric light chain were
expressed as described above for the humanized antibody.

This chimeric antibody is advantageous in that
it is characterized by activity substantially identical
5 to that of the native murine antibody, but contains
enough human sequences that it is anticipated to be
useful in human therapy.

Example 7 - ISI Assay

The Inhibition of Sporozoite Invasion assay is

10 performed as described in M. R. Hollingdale et al. ,
Immunol. . 132:909-913 (1984) to be used to assess
neutralizing effect against live P. falciparum
sporozoites. In the ISI assay, the human hepatoma cloned
cell line HepG2-A16 2 was grown to near confluency on 1%

15 C0 2 glass cover slips in MEM and 10% bovine fetal serum.
Antisera or purified antibodies were diluted in culture
medium (see table below) and added to the cell cultures.
30,000 P. falciparum sporozoites isolated from dissected
mosquito salivary glands are counted, diluted and added

2 0 to each cell culture. The cultures are incubated at 37 °C

for 2.5 hours, rinsed with PES, and fixed with methanol.
Fixed cultures are reacted in an immunoperoxidase
antibody assay using a labelled mAb which recognizes the
P. falciparum CS protein to visualize invaded
25 sporozoites. Then, the number of invading sporozoites

are counted by phase microscopy at 4 0 Ox. The ISI is the
percent reduction in invasion in the presence of the test
antibody, the humanized antibody, as compared to a
control (i.e. non-related) antibody. The assay ranks

3 0 antibodies in order according to their relative potency.

The following table provides the results
performed on the chimeric and synthetic antibodies
described previously. Values given are percent
inhibition and are the average of 2-3 independent assays.

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Summary

of ISI Studies

In fig/ml:

5.0 2.0

1.0

0.1

Chimeric

98.5

98 88

PfHzhc2-3/lcl-l

75.5

PfHzhc2-3/lcl-2

80.0

PfHzhc2-6/lcl-l

90.0

PfHzhc2-6/lcl-2

87.0

10 Numerous modifications and variations of the

present invention are included in the above-identified
specification and are expected to be obvious to one of
skill in the art. For example, recombinant antibodies
capable of neutralizing pathogens other than P.

15 falciparum may be provided according to the teachings of
this invention for the development of prophylactic agents
capable of conferring passive immunity to other human
diseases. Preferably, engineered antibodies capable of
recognizing repeat regions on other malaria pathogens or

2 0 engineered antibodies to any region on the surface of any
stages of the life-cycle of the Plasmodium species or
capable of neutralizing any stage in the life cycle of
the parasite, are desirable starting materials to develop
passive immunity agents according to this invention.

25 Such modifications and alterations to the compositions

and processes of the present invention are believed to be
encompassed in the scope of the claims appended hereto.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: SmithKline Beecham, Corporation

U. S. Government, Secretary of
the Navy

U. S. Government, Secretary of
the Army

(ii) TITLE OF INVENTION: Novel Antibodies for Conferring

Passive Immunity Against Infection by a
Pathogen in Man

(iii) NUMBER OF SEQUENCES: 61

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Howson and Howson

(B) STREET: Box 457, 321 Norristown Road

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19477

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/941,654

(B) FILING DATE: 09-SEP-1992

(viii) ATTORNEY /AGENT INFORMATION:

(A) NAME: Bak, Mary E.

(B) REGISTRATION NUMBER: 31,215

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (215) 540-9200

(B) TELEFAX: (215) 540-5818

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(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 164 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn
15 10 15

Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala

20 25 30

Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn

35 40 45

Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro

50 55 60

Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn

65 70 75

Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala

80 85 90

Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn

95 100 105

Ala Asn Pro
Asn Ala Asn
Pro Asn Ala
Asp Pro Asn

Asn Ala Asn
110

Pro Asn Ala
125

Asn Pro Asn
140

Val Asp Pro
155

Pro Asn Ala
Asn Pro Asn
Ala Asn Pro
Asn Val Asp

Asn Pro Asn
115

Ala Asn Pro
130

Asn Ala Asn
145

Pro Asn Val
160

Ala Asn Pro

120

Asn Ala Asn

135

Pro Asn Val

150

Asp Pro

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 163 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

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PCT/US93/08435

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro

15 10 15

Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn

20 25 30

Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala

35 40 45

Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn

50 55 60

Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro

65 70 75

Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn

80 85 90

Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala

95 100 105

Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn

110 115 120

Ala Asn Pro Asn Ala Asn Pro Asn Val Asp Pro Leu Arg Arg Thr

125 130 135

His Arg Gly Arg His His Arg Arg His Arg Cys Gly Cys Trp Arg

140 145 150

Leu Tyr Arg Arg His His Arg Trp Gly Arg Ser Gly Ser

155 160

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 339 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..339

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GAT ATT CAG CTG ACC CAG TCT CCA TCC TCC CTA GCT GTG TCA 42
Asp lie Gin Leu Thr Gin Ser Pro Ser Ser Leu Ala Val Ser
15 10

GTT GGA GAG AAG GTT ACT ATG AGC TGC AAG TCC AGT CAG AGC 84
Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gin Ser
15 20 25

CTT TTA TAT AGT AGC AAT CAA AAG AAT TAC TTG GCC TGG TAC 12 6

Leu Leu Tyr Ser Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr

30 35 40

CAG CAG AAA CCA GGG CAG TCT CCT AAA CTG CTG ATT TAC TGG 168
Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu lie Tyr Trp

45 50 55

GCA TCC ACT AGG GAA TCT GGG GTC CCT GAT CGC TTC ACA GGC 210
Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly

60 65 70

AGA GGA TCC GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT GTG 252
Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Val

75 80

AAG GCT GAA GAC CTG GCA GTT TAT TAC TGT CAG CAA TAT TAT 294
Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr
85 90 95

AGC TAT CCT CGG ACG TTC GGT GGA " GGG ACC AAG CTG GAG ATC 33 6

Ser Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu lie
100 105 110

AAA
Lys

339

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Asp lie Gin Leu Thr Gin Ser Pro Ser Ser Leu Ala Val Ser Val
15 10 15

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Gly Glu Lys
Tyr Ser Ser
Pro Gly Gin
Glu Ser Gly
Asp Phe Thr
Val Tyr Tyr
Gly Gly Thr

Val Thr Met
20

Asn Gin Lys
35

Ser Pro Lys
50

Val Pro Asp
65

Leu Thr lie
80

Cys Gin Gin
95

Lys Leu Glu
110

Ser Cys Lys
Asn Tyr Leu
Leu Leu lie
Arg Phe Thr
Ser Ser Val
Tyr Tyr Ser
lie Lys

Ser Ser Gin
25

Ala Trp Tyr
40

Tyr Trp Ala
55

Gly Arg Gly
70

Lys Ala Glu
85

Tyr Pro Arg
100

Ser Leu Leu

Gin Gin Lys

Ser Thr Arg

Ser Gly Thr

Asp Leu Ala

Thr Phe Gly

105

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 339 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME /KEY: CDS

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GAT ATC GTG ATG ACC CAG TCT CCA GAC TCG CTA GCT GTG TCT 42
Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser
15 10

CTG GGC GAG AGG GCC ACC ATC AAC TGC AAG AGC TCT CAG AGC 84
Leu Gly Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser
15 20 25

CTT TTA TAC TCG AGC AAT CAA AAG AAT TAC TTG GCC TGG TAT 126
Leu Leu Tyr Ser Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr
30 35 40

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CAG CAG AAA CCC GGG CAG TCT CCT AAG TTG CTC ATT TAC TGG 168

Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu lie Tyr Trp

45 50 55

GCG TCA ACT AGG GAA TCT GGG GTA CCT GAC CGA TTC AGT GGC 210
Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

60 65 70

AGC GGG TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGC CTG 252
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu

75 80

CAG GCT GAA GAT GTG GCA GTA TAC TAC TGT CAG CAA TAT TAT 294

Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr

85 90 95

AGC TAT CCG CGG ACG TTC GGC GGA GGG ACC AAG GTG GAG ATC 33 6
Ser Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu lie
100 105 110

AAA
Lys

339

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Asp lie Val
1

Gly Glu Arg
Tyr Ser Ser
Pro Gly Gin

Met Thr Gin
5

Ala Thr lie
20

Asn Gin Lys

Ser Pro Lys
50

Ser Pro Asp
Asn Cys Lys
Asn Tyr Leu
Leu Leu lie

Ser Leu Ala
10

Ser Ser Gin
25

Ala Trp Tyr
40

Tyr Trp Ala
55

Val Ser Leu

Ser Leu Leu

Gin Gin Lys

Ser Thr Arg

Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

65 70 75

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Asp Phe Thr
Val Tyr Tyr
Gly Gly Thr

Leu Thr lie
80

Cys Gin Gin
95

Lys Val Glu
110

Ser Ser Leu
Tyr Tyr Ser
lie Lys

Gin Ala Glu
85

Tyr Pro Arg
100

Asp Val Ala

Thr Phe Gly

105

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 339 base pairs

(B) TYPE: nucleic acid

( C ) STRANDEDNESS : double

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME /KEY: CDS

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GAT ATC GTG ATG ACC CAG TCT CCA GAC TCG CTA GCT GTG TCT 42

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser
15 10

CTG GGC GAG AGG GCC ACC ATC* AAC*TGC AAG AGC TCT CAG AGC 84

Leu Gly Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser
15 20 25

CTT TTA TAC TCG AGC AAT CAA AAG AAT TAC TTG GCC TGG TAT 126
Leu Leu Tyr Ser Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr
30 35 40

CAG CAG AAA CCC GGG CAG CCT CCT AAG TTG CTC ATT TAC TGG 168

Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu lie Tyr Trp

45 50 55

GCG TCG ACT AGG GAA TCT GGG GTA CCT GAC CGA TTC AGT GGC 210

Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

60 65 70

AGC GGG TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGC CTG 252
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu

75 80

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CAG GCT GAA GAT GTG GCA GTA TAG TAC TGT CAG CAA TAT TAT 294
Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr
85 90 95

AGC TAT CCG CGG ACG TTC GGC GGA GGG ACC AAG GTG GAG ATC 336
Ser Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu lie
100 105 110

AAA 339
Lys

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu
15 10 15

Gly Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Leu Leu

20 25 30

Tyr Ser Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys

35 40 45

Pro Gly Gin Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr Arg

50 55 60

Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

65 70 75

Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Ala Glu Asp Val Ala

80 85 90

Val Tyr Tyr Cys Gin Gin Tyr Tyr Ser Tyr Pro Arg Thr Phe Gly

95 100 105

Gly Gly Thr Lys Val Glu lie Lys

110

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(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 354 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME /KEY: CDS

(B) LOCATION: 1..354

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CTC GAG TCT GGG GGA GGC TTA GTG AAG CCT GGA GGG TCC CTG 42
Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu
15 10

AAA ATC TCC TGC GCA GCC TCT GGA TTC ACT TTC AGT AGC TAT 84
Lys lie Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
15 20 25

GCC ATG TCT TGG GTT CGC CAG TCT CCA GAG AAG AGG CTG GAG 12 6
Ala Met Ser Trp Val Arg Gin Ser Pro Glu Lys Arg Leu Glu
30 35 40

TGG GTC GCA GAA ATT AGT GAT GGT GGT AGT TAC ACC TAC TAT 168
Trp Val Ala Glu lie Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr

45 50 55

CCA GAC ACT GTG ACG GGC CGA TTC ACC ATC TCC AGA GAC AAT 210
Pro Asp Thr Val Thr Gly Arg Phe Thr lie Ser Arg Asp Asn

60 65 70

GCC AAG AAC ACC CTA TAC CTG GAA ATG AGC AGT CTG AGG TCT 252
Ala Lys Asn Thr Leu Tyr Leu Glu Met Ser Ser Leu Arg Ser

75 80

GAG GAC ACG GCC ATG TAT TAC TGT GCA AGC CTC ATC TAC TAT 2 94
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ser Leu lie Tyr Tyr
85 90 95

GGT TAC GAC GGG TAT GCT ATG GAC TAC TGG GGT CAA GGA ACC 336
Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr
100 105 " 110

TCA GTC ACC GTC TCC TCA
Ser Val Thr Val Ser Ser

115

354

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(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys
1 5 10 15

lie Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met

20 25 30

Ser Trp Val Arg Gin Ser Pro Glu Lys Arg Leu Glu Trp Val Ala

35 40 45

Glu lie Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Leu

65 70 75

Tyr Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr

80 85 90

Tyr Cys Ala Ser Leu lie Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met

95 100 105

Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser

110 115

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 389 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..366

WO 94/05690

PCT/US93/08435

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GAG GTG CAG CTG CTC GAG TCT GGG GGA GGC TTG GTA CAG CCT 42

Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro

15 10

GGG GGG TCC CTG AGA CTC TCC TGT GCA GCT AGC GGA TTC ACC 84

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

15 20 25

TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG 12 6

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gin Ala Pro Gly

30 35 40

AAA GGT CTA GAG TGG GTC TCA GAA ATT AGT GAT GGT GGT AGT 168

Lys Gly Leu Glu Trp Val Ser Glu lie Ser Asp Gly Gly Ser

^ 45 50

TAC ACC TAC TAT CCA GAC ACT GTG ACG GGC CGG TTC ACG ATA 210
Tyr Thr Tyr Tyr Pro Asp Thr Val Thr Gly Arg Phe Thr lie

60 65 70

TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC 252
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn

75 80

AGC CTG AGA GCC GAG GAC ACT GCA GTA TAT TAC TGT GCG AAA 294
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys
85 90 95

CTC ATC TAC TAT GGT TAC GAC GGG TAT GCT ATG GAC TAC TGG 336
Leu lie Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp
100 105 110

GGC CAG GGT ACC CTG GTC ACC GTG AGC TCA GCTAGTACCA 376
Gly Gin Gly Thr Leu Val Thr Val Ser Ser

115 120

AGGGCCCAAG CTT 3 89

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 122 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly
1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ser Glu lie Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr

50 55 60

Pro Asp Thr Val Thr Gly Arg Phe Thr lie Ser Arg Asp Asn Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Lys Leu lie Tyr Tyr Gly Tyr Asp

95 100 105

Gly Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val

110 115 120

Ser Ser

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 389 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME /KEY: CDS

(B) LOCATION: 1..366

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

GAG GTG CAG CTG CTC GAG TCT GGG GGA GGC TTG GTA CAG CCT 42
Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro
15 10

GGG GGG TCC CTG AGA CTC TCC TGT GCA GCT AGC GGA TTC ACC 84
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
15 20 25

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TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG 12 6

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gin Ala Pro Gly
30 35 40

AAA GGT CTA GAG TGG GTC TCA GAA ATT AGT GAT GGT GGT AGT 168

Lys Gly Leu Glu Trp Val Ser Glu lie Ser Asp Gly Gly Ser

45 50 55

TAC ACC TAC TAT CCA GAC ACT GTG ACG GGC CGG TTC ACG ATA 210
Tyr Thr Tyr Tyr Pro Asp Thr Val Thr Gly Arg Phe Thr lie

60 65 70

TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC 252

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn

75 80

AGC CTG AGA GCC GAG GAC ACT GCA GTG TAT TAC TGT GCA TCT 294
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser
85 90 95

CTC ATC TAC TAT GGT TAC GAC GGG TAT GCT ATG GAC TAC TGG 336
Leu lie Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp
100 105 110

GGC CAA GGT ACC CTG GTC ACC GTG AGC TCA GCTAGTACCA 376
Gly Gin Gly Thr Leu Val Thr Val Ser Ser

115 120

AGGGCCCAAG CTT 389

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 122 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly
15 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu

35 40 45

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Glu Trp Val
Pro Asp Thr
Lys Asn Thr
Thr Ala Val
Gly Tyr Ala
Ser Ser

Ser Glu lie
50

Val Thr Gly
65

Leu Tyr Leu
80

Tyr Tyr Cys

Met Asp Tyr
110

Ser Asp Gly
Arg Phe Thr
Gin Met Asn
Ala Ser Leu
Trp Gly Gin

Gly Ser Tyr
55

lie Ser Arg
70

Ser Leu Arg
85

lie Tyr Tyr
100

Gly Thr Leu
115

Thr Tyr Tyr

Asp Asn Ser

Ala Glu Asp

Gly Tyr Asp

105

Val Thr Val

120

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
AGCTATGCCA TGAGC 15

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Ser Tyr Ala Met Ser
1 5

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(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

( C ) STRANDEDNESS : double

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GAAATTAGTG ATGGTGGTAG TTACACCTAC TATCCAGACA CTGTGACGGG C 51

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

Glu lie Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr

1 k 5 10

Val Thr Gly
15

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
CTCATCTACT ATGGTTACGA CGGGTATGCT ATGGACTAC 39

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(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Leu lie Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr
1 5 ~ 10

(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

AAGAGCTCTC AGAGCCTTTT ATACTCGAGC AATCAAAAGA ATTACTTGGC C 51

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Lys Ser Ser Gin Ser Leu Leu Tyr Ser Ser Asn Gin Lys Asn
15 10

Tyr Leu Ala
15

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(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23
TGGGCGTCAA CTAGGGAATC T

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24

Trp Ala Ser Thr Arg Glu Ser
1 5

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25
CAGCAATATT ATAGCTATCC GCGGACG

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

WO 94/05690

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26

Gin Gin Tyr Tyr Ser Tyr Pro Arg Thr
1 5

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27

Pro Asn Ala Asn Pro Asn
1 5

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28
CCAGATGTAA GCTTCAGCTG ACCCAGTCTC CA

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
CATCTAGATG GCGCCGCCAC AGTACGTTTG ATCTCCAGCT TGGTCCC 47

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
GGGGTACCAG GTCCARCTKC TCGAGTCWGG 3 0

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
GCCTGCAGCT AGCTGAGGAG ACGGTGACCG TGGTCCCTTG GCCCCAG 47

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
AGCTATGCCA TGTCT 15

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(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

Ser Tyr Ala Met Ser
1 * 5

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
AAGTCCAGTC AGAGCCTTTT ATATAGTAGC AATCAAAAGA ATTACTTGGC C 51

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

Lys Ser Ser Gin Ser Leu Leu Tyr Ser Ser Asn Gin Lys Asn
1 5 10

Tyr Leu Ala
15

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(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36
TGGGCATCCA CTAGGGAATC T

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37

Trp Ala Ser Thr Arg Glu Ser
1 5

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38
CAGCAATATT ATAGCTATCC TCGGACG

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(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39

Gin Gin Tyr Tyr Ser Tyr Pro Arg Thr
1 5

(2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40
TGGGCGTCGA CTAGGGAATC T

(2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41

Trp Ala Ser Thr Arg Glu Ser
1 5

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(2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3 66 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..366

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

GAG GTG CAG CTG CTC GAG TCT GGG GGA GGC TTG GTA CAG CCT 42
Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro
15 10

GGG GGG TCC CTG AGA CTC TCC TGT GCA GCT AGC GGA TTC ACC 84

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
15 20 25

TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG 126

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gin Ala Pro Gly
30 35 40

AAA GGT CTA GAG TGG GTC GCA GAG ATC TCT GAT GGT GGT AGT 168
Lys Gly Leu Glu Trp Val Ala Glu lie Ser Asp Gly Gly Ser

45 50 55

TAC ACC TAC TAT CCA GAC ACT GTG ACG GGC CGG TTC ACG ATA 210

Tyr Thr Tyr Tyr Pro Asp Thr Val Thr Gly Arg Phe Thr lie

60 65 70

TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC 252

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn

75 80

AGC CTG AGA GCC GAG GAC ACT GCA GTG TAT TAC TGT GCA TCT 294

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser
85 90 95

CTC ATC TAC TAT GGT TAC GAC GGG TAT GCT ATG GAC TAC TGG 336

Leu lie Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp
100 105 110

GGC CAA GGT ACC CTG GTC ACC GTG AGC TCA

Gly Gin Gly Thr Leu Val Thr Val Ser Ser

115 120

366

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(2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 122 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly
15 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ala Glu lie Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr

50 55 60

Pro Asp Thr Val Thr Gly Arg Phe Thr lie Ser Arg Asp Asn Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Ser Leu lie Tyr Tyr Gly Tyr Asp

95 100 105

Gly Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val

110 115 120

Ser Ser

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
TAGTGAAGAA TTCGAGGACG CCAGCAACAT GGTGTTGCAG ACCCAGGTCT 50
TCATTTCTCT GTTGCTCTGG ATCTCTGGTG CCTACGGGGA GGTGCAG 97

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 164 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

GCTAGCGGAT TCACCTTTAG CAGCCATGTC GGACCCCCCA GGGACTCTGA 50

GAGGACACGT CGATCGCCTA AGTGGAAATC CTATGCCATG AGCTGGGTCC 100

GCCAGGCTCC AGGGAAAGGT CTAGAGTGGG TCTCAGAAAT CTTTATAGTG 150

ATGGTGGTAG TTAC 164

(2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 164 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

GAACACGCTG TATCTGCAAA TGAACAGCCT GAGAGCCGAG GACACGTCTC 50

TGTTAAGGTT CTTGTGCGAC ATAGACGTTT ACTGCAGTAT ATTACTGTGC 100

GAAACTCATC TACTATGGTT ACGACGGGTA TGCTATGGAC TAGCTGCCCA 150

TACGATACCT GATC 164

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(2) INFORMATION FOR SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 85 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
TTCTTGAAAG CTTGGGCCCT TGGTACTAGC TGAGCTCACG GTGACCAGGG 50
TACCCTGGCC CCAGTAGTCC ATAGCATACC CGTCG 85

(2) INFORMATION FOR SEQ ID NO: 48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
CATTTGCAGA TACAGCGTGT TCTTGGAATT GTCTCTGGAT ATCGTGAACC 50
GGCCCGTCAC AGTGTCTGGA TAGTAGGTGT AACTACCACC ATCACTAATT TC 102

(2) INFORMATION FOR SEQ ID NO: 49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 101 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
CTAAAGGTGA ATCCGCTAGC TGCACAGGAG AGTCTCAGGG ACCCCCCAGG 50
CTGTACCAAG CCTCCCCCAG ACTCGAGCAG CTGCACCTCC CCGTAGGCAC C 101

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(2) INFORMATION FOR SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
CCGCGAATTC GAGGACGCCA GCAAC 25

(2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
CCGCAAGCTT GGGCCCTTGG TACTAGCT 28

(2) INFORMATION FOR SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
TAAGCGGAAT TCGTAGTCGG ATATCGTGAT GACCCAGTCT CCAGACTCGC 50
TAGCTGTGTC TCTGGGCGAG AGGGC 75

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(2) INFORMATION FOR SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 90 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
TTACTTGGCC TGGTATCAGC AGAAACCCGG GCAGTCTCCT AAGTTGCTCA 50
TAGTTTTCTT AATGAACCGG ACTTACTGGG CGTCAACTAG 90

(2) INFORMATION FOR SEQ ID NO: 54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 93 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
GACAGATTTC ACTCTCACCA TCAGCAGCCT GCAGGCTGAA GATGTGGCAG 50
TATACTACTG CTGTCTAAAG TGTCAGCAAT ATTATAGCTA TCC 93

(2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 86 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
CAGTTGGAAA GCTTGGCGCC GCCACAGTAC GTTTGATCTC CACCTTGGTC 50
CCTCCGCCGA ACGTCCGCGG ATAGCTATAA TATTGC 86

(2) INFORMATION FOR SEQ ID NO: 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
GTGAAATCTG TCCCAGACCC GCTGCCACTG AATCGGTCAG GTACCCCAGA 50
TTCCCTAGTT GACGCC 66

(2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
CAGGCCAAGT AATTCTTTTG ATTGCTCGAG TATAAAAGGC TCTGAGAGCT 50
CTTGCAGTTG ATGGTGGCCC TCTCGCCC 78

(2) INFORMATION FOR SEQ ID NO: 58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

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(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
GCGGAATTCG TAGTCGGATA TCGTGATGAC 30

(2) INFORMATION FOR SEQ ID NO: 59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
TGGAAAGCTT GGCGCCGCCA CAGTACGTTT GATC 34

(2) INFORMATION FOR SEQ ID NO: 60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME /KEY : CDS

(B) LOCATION: 1..57

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:

ATG GTG TTG CAG ACC CAG GTC TTC ATT TCT CTG TTG CTC TGG 42
Met Val Leu Gin Thr Gin Val Phe lie Ser Leu Leu Leu Trp
15 10

ATC TCT GGT GCC TAC 57
lie Ser Gly Ala Tyr
15

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(2) INFORMATION FOR SEQ ID NO: 61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

Met Val Leu Gin Thr Gin Val Phe lie Ser Leu Leu Leu Trp lie
15 10 15

Ser Gly Ala Tyr

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WHAT IS CLAIMED IS:

1. A fusion molecule comprising a first
fusion partner nucleotide sequence encoding an amino acid
sequence having the antigen specificity of a anti-
Plasmodium antibody, operatively linked in frame to a
second fusion partner nucleotide sequence.

2. The molecule according to claim 1 wherein
said first fusion partner is a synthetic immunoglobulin
variable region nucleotide sequence encoding an amino
acid sequence comprising a complementarity determining
region originating from a Plasmodium antibody, a fragment
or allelic variation or modification thereof.

3. The molecule according to claim 2 wherein
said second fusion partner is a heterologous
immunoglobulin variable framework region.

4. The molecule according to claim 2 wherein
said variable region nucleotide sequence is selected from
the group consisting of a heavy chain variable region and
a light chain variable region.

5. The molecule according to claim 4 selected
from the group consisting of

(a) a heavy chain nucleotide sequence of

Fig. 5 (SEQ ID NO: 11);

(b) a heavy chain nucleotide sequence of

Fig. 6 (SEQ ID NO: 13) ;

(d) a light chain nucleotide sequence of
Fig. 3 (SEQ ID NO: 7); and

(e) functional fragments thereof.

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6. The molecule according to claim 4 wherein
said first fusion partner nucleotide sequence comprises a
sequence selected from the group consisting of

(a) AGCTATGCCATGAGC : SEQ ID NO: 15;

( b ) GAAATTAGTGATGGTGGTAGTTACACCTACTATCCA

GACACTGTGACGGGC : SEQ ID NO: 17;
( c ) CTCATCTACTATGGTTACGACGGGTATGCTATGGAC

TAC: SEQ ID NO: 19;
( d ) AAGAGCTCTCAGAGCCTTTTATACTCGAGCAATCAA

AAGAATTACTTGGCC: SEQ ID NO: 21;

(e) TGGGCGTCAACTAGGGAATCT : SEQ ID NO: 23;

(f) CAGCAATATTATAGCTATCCGCGGACG : SEQ ID
NO: 25;

(g) AGCTATGCCATGTCT : SEQ ID NO: 32;

( h ) AAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAA
AGAATTACTTGGCC : SEQ ID NO: 34;

(i) TGGGCATCCACTAGGGAATCT : SEQ ID NO: 36;
(j) CAGCAATATTATAGCTATCCTCGGACG : SEQ ID

NO: 38;

(k) TGGGCGTCGACTAGGGAATCT: SEQ ID NO: 41;
and an allelic variation or modification thereof,
characterized by the antigen specificity of murine NFS2 ,
said nucleic acid sequence optionally containing
restriction sites to facilitate insertion into a desired
antibody framework region or a plasmid vector.

7. A synthetic immunoglobulin variable region
nucleotide sequence encoding an amino acid sequence
comprising a complementarity determining region
originating from a Plasmodium antibody, a fragment or
allelic variation or modification thereof.

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8. A fusion protein comprising a first amino
acid sequence derived from a Plasmodium antibody capable
of binding an epitope on a selected Plasmodium species,
said sequence having the antigen specificity of said
antibody fused to a heterologous second amino acid
sequence .

9. The fusion protein according to claim 8
wherein said first amino acid sequence comprises an amino
acid sequence selected from the group consisting of:

(a) a variable heavy chain sequence of said

antibody;

(b) a variable light chain sequence of said

antibody;

(d) a functional fragment of (a) through (c) *

10. The fusion protein according to claim 8
wherein said fusion protein is selected from the group
consisting of

(a) a complete engineered antibody, having
full length heavy and light chains comprising at least
fragments of the variable regions derived from said
Plasmodium antibody;

(b) the F ab or (F ab ') 2 fragment of the
engineered antibody of (a) ;

(d) an F v fragment of the engineered antibody
of (a) ; and

(e) a single-chain antibody derived from the
engineered antibody of (a) ;

said protein having the same specificity as
said Plasmodium antibody.

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11. An engineered P. falciparum antibody
comprising a heavy chain comprising a complementarity
determining region derived from the variable heavy chain
region of a non-human P. falciparum monoclonal antibody.

12. The antibody according to claim 11 wherein
said non-human CDRs are in operative association with one

of the group consisting of

(a) a selected human antibody heavy chain

framework and constant regions; and

(b) the heavy chain framework from said
antibody and a constant region from a selected human
antibody.

13 . The antibody according to claim 11 further
comprising a light chain selected from the group
consisting of

(a) a light chain comprising a CDR derived
from the variable light chain region of said monoclonal
antibody in operative association with selected human
antibody light chain framework and constant regions;

(b) the light chain framework from said
antibody and a constant region from a selected human
antibody;

(d) the complete light chain from a
selected human antibody.

14. The antibody according to claim 11 ,
wherein said heavy chain comprises a variable heavy chain
sequence selected from the sequences of Fig. 5 (SEQ ID
NO: 12), Fig. 6 (SEQ ID NO: 14), Pfhzhc2-3 (SEQ ID
NO:14), and Pfhzhc2-6 (SEQ ID N0:42).

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15. The antibody according to claim 13 wherein
said light chain comprises a variable light chain
sequence selected from the sequences of Fig. 2 (SEQ ID
NO: 6) and Fig. 3 (SEQ ID NO: 8).

16. The antibody according to claim 13 wherein
light chain complementarity determining region is
selected from one or more of the sequences consisting of

(a) LysSerSerGlnSerLeuLeuTyrSerSerAsn
GlnLysAsnTyrLeuAla : SEQ ID NO: 22;

(b) TrpAlaSerThrArgGluSer : SEQ ID NO: 24;
and

17. The antibody according to claim 11 wherein
said heavy chain complementarity determining region is
selected from one or more of the sequences consisting of

(a) SerTyrAlaMetSer: SEQ ID NO: 16;

(b) GluIleSerAspGlyGlySerTyrThrTyrTyrPro
AspThrValThrGly: SEQ ID NO: 18; and

18. An anti-P. falciparum complementarity
determining region peptide selected from the group
consisting of

(a) SerTyrAlaMetSer: SEQ ID NO: 16;

(b) GluIleSerAspGlyGlySerTyrThrTyrTyr
ProAspThrValThrGly: SEQ ID NO: 18;

( d ) Ly sSer SerG InSerLeuLeuTyr Ser Ser Asn
GlnLysAsnTyrLeuAla: SEQ ID NO: 22;

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(e) TrpAlaSerThrArgGluSer: SEQ ID NO: 24;

(f) GlnGlnTyrTyrSerTyrProArgThr : SEQ ID
NO: 26;

and an analog thereof, characterized by the antigen
specificity of NFS2 .

19. A synthetic immunoglobulin variable chain
amino acid sequence comprising a complementarity
determining region originating from a Plasmodium antibody
in a heterologous variable chain framework, a fragment or
analog thereof sharing the ant i -Plasmodium antigen
specificity of said sequence.

20. The sequence according to claim 19
selected from the group consisting of the amino acid
sequences of Fig. 5 (SEQ ID NO: 12) , Fig. 6 (SEQ ID NO:
14), Fig. 2 (SEQ ID NO: 6) and Fig. 3 (SEQ ID NO: 8).

21. A monoclonal antibody, other than NFS2,
which is capable of binding to a P. falciparum epitope
comprising the sequence Pro Asn Ala Asn Pro Asn SEQ ID
NO: 27, a F ab fragment thereof, or a (F ab ')2 fragment
thereof .

22. A pharmaceutical prophylactic composition
comprising a fusion protein or antibody according to any
of claims 8 through 21 and a pharmaceutically acceptable
carrier or diluent.

23. A pharmaceutical composition according to
claim 22 wherein said protein is a humanized P.
falciparum antibody.

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24. A recombinant plasmid comprising a nucleic
acid sequence of any of claims 1 through 7 in operative
association with a regulatory control sequence capable of
directing the replication and expression of said nucleic
acid sequence in a selected host cell.

25. A mammalian cell line transfected with at
least one recombinant plasmid comprising a nucleic acid
sequence of any of claims 1 through 7.

26. A method of producing a engineered
antibody comprising culturing a mammalian cell line
transfected with at least one recombinant plasmid
comprising a nucleic acid sequence of any of claims 1
through 7 under suitable conditions permitting expression
and assembly of complementary heavy and light chains, and
recovering the assembled antibody from the cell culture.

27. The use of a protein or antibody of claims
8 through 21 in the preparation of a pharmaceutical
composition suitable for passively protecting a human
against infection by a Plasmodium species.

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FIGURE 1

GAT ATT CAG CTG ACC CAG TCT CCA TCC TCC CTA 33
Asp lie Gin Leu Thr Gin Ser Pro Ser Ser Leu
15 10

GCT GTG TCA GTT GGA GAG AAG GTT ACT ATG AGC 66
Ala Val Ser Val Gly Glu Lys Val Thr Met Ser

15 20

TGC AAG TCC AGT CAG AGC CTT TTA TAT AGT AGC 99

Cys Lys Ser Ser Gin Ser Leu Leu Tvr Ser Ser

25 30

AAT CAA AAG AAT TAC TTG GCC TGG TAC CAG CAG 132
Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin
35 40

AAA CCA GGG CAG TCT CCT AAA CTG CTG ATT TAC 165

Lys Pro Gly Gin Ser Pro Lys Leu Leu lie Tyr
45 50 55

TGG GCA TCC ACT AGG GAA TCT GGG GTC CCT GAT 198
Trp Ala Ser Thr Ara Glu Ser Gly Val Pro Asp

60 65

CGC TTC ACA GGC AGA GGA TCC GGG ACA GAT TTC 231
Arg Phe Thr Gly Arg Gly Ser Gly Thr Asp Phe

70 75

ACT CTC ACC ATC AGC AGT GTG AAG GCT GAA GAC 264
Thr Leu Thr lie Ser Ser Val Lys Ala Glu Asp

80 * 85

CTG GCA GTT TAT TAC TGT CAG CAA TAT TAT AGC 297

Leu Ala Val Tyr Tyr Cys Gin Gin Tvr Tvr Ser
90 95

TAT CCT CGG ACG TTC GGT GGA GGG ACC AAG CTG 330

Tyr Pro Ara Thr Phe Gly Gly Gly Thr Lys Leu
100 105 110

GAG ATC AAA 339
Glu lie Lys

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Figure 2

Eco RV Nhe I

GAT ATC GTG ATG ACC CAG TCT CCA GAC TCG CTA GCT GTG 39

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val
15 10

Sst I

TCT CTG GGC GAG AGG GCC ACC ATC AAC TGC AAG AGC TCT 78
Ser Leu Gly Glu Arg Ala Thr lie Asn Cys Lys Ser Ser
15 20 25

Xho I

CAG AGC CTT TTA TAC TCG AGC AAT CAA AAG AAT TAC TTG 117
Gin Ser Leu Leu Tyr Ser Ser Asn Gin Lvs Asn Tvr Leu

30 35

Sma I

GCC TGG TAT CAG CAG AAA CCC GGG CAG TCT CCT AAG TTG 156
Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu
40 45 50

Hinc 11 Kon 1

CTC ATT TAC TGG GCG TCA ACT AGG GAA TCT GGG GTA CCT 195
Leu lie Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro

55 60 65

GAC CGA TTC AGT GGC AGC GGG TCT GGG ACA GAT TTC ACT 234
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

70 75

Pst 1 Ace
CTC ACC ATC AGC AGC CTG CAG GCT GAA GAT GTG GCA GTA 273
Leu Thr lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val
80 85 90

I Sst II

TAC TAC TGT CAG CAA TAT TAT AGC TAT CCG CGG ACG TTC 312
Tyr Tyr Cys Gin Gin Tvr Tyr Ser Tyr Pro Arg Thr Phe

95 100

Stv I

GGC GGA GGG ACC AAG GTG GAG ATC AAA 339

Gly Gly Gly Thr Lys Val Glu lie Lys
105 110

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Figure 3

Eco RV Nhe I

GAT ATC GTG ATG ACC CAG TCT CCA GAC TCG CTA GCT GTG 3 9

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val
1 5 10

Sst I

TCT CTG GGC GAG AGG GCC ACC ATC AAC TGC AAG AGC TCT 78
Ser Leu Gly Glu Arg Ala Thr lie Asn Cys Lys Ser Ser
15 20 25

Xhol

CAG AGC CTT TTA TAC TCG AGC AAT CAA AAG AAT TAC TTG 117
Gin Ser Leu Leu Tyr Ser Ser Asn Gin Lys Asn Tyr Leu

30 35

Sma I

GCC TGG TAT CAG CAG AAA CCC GGG CAG CCT CCT AAG TTG 156
Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu
40 45 50

Hinc II Kon I

CTC ATT TAC TGG GCG TCG ACT AGG GAA TCT GGG GTA CCT 195
Leu lie Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro

55 60 65

GAC CGA TTC AGT GGC AGC GGG TCT GGG ACA GAT TTC ACT 234
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

70 - 75

Pst I Acc

CTC ACC ATC AGC AGC CTG CAG GCT GAA GAT GTG GCA GTA 273

Leu Thr lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val
80 85 90

T Sst II

TAC TAC TGT CAG CAA TAT TAT AGC TAT CCG CGG ACG TTC 312
Tyr Tyr Cys Gin Gin Tvr Tvr Ser Tyr Pro Arg Thr Phe

95 100

Stv I

GGC GGA GGG ACC AAG GTG GAG ATC AAA 339
Gly Gly Gly Thr Lys Val Glu lie Lys
105 ~ 110

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PCT/US93/08435

4/9

FIGURE 4

CTC GAG TCT GGG GGA GGC TTA GTG AAG CCT 30
Leu Glu Ser Gly Gly Gly Leu Val Lys Pro
15 10

GGA GGG TCC CTG AAA ATC TCC TGC GCA GCC 60
Gly Gly Ser Leu Lys lie Ser Cys Ala Ala

15 20

TCT GGA TTC ACT TTC AGT AGC TAT GCC ATG 90

Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met

25 30

TCT TGG GTT CGC CAG TCT CCA GAG AAG AGG 120
Ser Trp Val Arg Gin Ser Pro Glu Lys Arg

35 40

CTG GAG TGG GTC GCA GAA ATT AGT GAT GGT 150
Leu Glu Trp Val Ala Glu lie Ser Asp Glv

45 50

GGT AGT TAC ACC TAC TAT CCA GAC ACT GTG 180
Gly Ser Tyr Thr Tvr Tyr Pro Asp Thr Val

55 60

ACG GGC CGA TTC ACC ATC TCC AGA GAC AAT 210
Thr Glv Arg Phe Thr lie Ser Arg Asp Asn

65 70

GCC AAG AAC ACC CTA TAC CTG GAA ATG AGC 240

Ala Lys Asn Thr Leu Tyr Leu Glu Met Ser

75 80

AGT CTG AGG TCT GAG GAC ACG GCC ATG TAT 270
Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr

85 90

TAC TGT GCA AGC CTC ATC TAC TAT GGT TAC 300
Tyr Cys Ala Ser Leu lie Tyr Tvr Glv Tvr

95 100

GAC GGG TAT GCT ATG GAC TAC TGG GGT CAA 330
Asp Glv Tvr Ala Met Asp Tvr Trp Gly Gin

105 110

GGA ACC TCA GTC ACC GTC TCC TCA
Gly Thr Ser Val Thr Val Ser Ser

115

354

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PCT/US93/08435

Figure 5

Xho I

GAG GTG CAG CTG CTC GAG TCT GGG GGA GGC TTG GTA CAG 39

Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin
15 10

Nhe I

CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCT AGC GGA 78
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
15 20 25

TTC ACC TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG 117
Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gin

30 35

Xba I

GCT CCA GGG AAA GGT CTA GAG TGG GTC TCA GAA ATT AGT 156
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Glu lie Ser
40 45 50

GAT GGT GGT AGT TAC ACC TAC TAT CCA GAC ACT GTG ACG 195
Asp Glv Glv Ser Tvr Thr Tvr Tyr Pro Asp Thr Val Thr

55 60 65

Eco RV

GGC CGG TTC ACG ATA TCC AGA GAC AAT TCC AAG AAC ACG 234
Glv Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr

70 75

CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACT 273

Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr
80 85 90

Pst I

GCA GTA TAT TAC TGT GCG AAA CTC ATC TAC TAT GGT TAC 312
Ala Val Tyr Tyr Cys Ala Lys Leu lie Tyr Tyr Gly Tyr

95 100

Kpn I

GAC GGG TAT GCT ATG GAC TAC TGG GGC CAG GGT ACC CTG 3 51
Asp Glv Tvr Ala Met Asp Tvr Trp Gly Gin Gly Thr Leu
105 110 115

Sst I

GTC ACC GTG AGC TCA GCTAGTACCA AGGGCCCAAG CTT 389

Val Thr Val Ser Ser

120

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Figure 6

Xho I

GAG GTG CAG CTG CTC GAG TCT GGG GGA GGC TTG GTA CAG 39
Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin
15 10

Nhe I

CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCT AGC GGA 78
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
15 20 25

TTC ACC TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG 117
Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gin

30 35

Xba I

GCT CCA GGG AAA GGT CTA GAG TGG GTC TCA GAA ATT AGT 156
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Glu lie Ser
40 45 50

GAT GGT GGT AGT TAC ACC TAC TAT CCA GAC ACT GTG ACG 195
Asp Gly Glv Ser Tyr Thr Tvr Tyr Pro Asp Thr Val Thr

55 60 65

EcoRV

GGC CGG TTC ACG ATA TCC AGA GAC AAT TCC AAG AAC ACG 2 34
Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr
~~ v 70 " 75

CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACT 273
Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr
80 85 90

Pst I

GCA GTG TAT TAC TGT GCA TCT CTC ATC TAC TAT GGT TAC 312

Ala Val Tyr Tyr Cys Ala Ser Leu lie Tvr Tvr Gly Ty r

95 100

Kpn I

GAC GGG TAT GCT ATG GAC TAC TGG GGC CAA GGT ACC CTG 351
Asp Glv Tyr Ala Met Asp Tvr Trp Gly Gin Gly Thr Leu
105 110 115

Sst I

GTC ACC GTG AGC TCA GCTAGTACCA AGGGCCCAAG CTT 389
Val Thr Val Ser Ser

120

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PCT/US93/08435

SUBSTITUTE SHEET

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8/9

i — »

LL_

SUBSTITUTE SHEET

WO 94/05690

9/9

PCT/US93/08435

FIGURE 9

GAG GTG CAG CTG CTC GAG TCT GGG GGA GGC TTG GTA CAG 39
Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin
15 10

CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCT AGC GGA 78
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
15 20 25

TTC ACC TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG 117
Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gin

30 35

GCT CCA GGG AAA GGT CTA GAG TGG GTC GCA GAG ATC TCT 156

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Glu lie Ser
40 45 50

GAT GGT GGT AGT TAC ACC TAC TAT CCA GAC ACT GTG ACG 195
Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val Thr

55 60 65

GGC CGG TTC ACG ATA TCC AGA GAC AAT TCC AAG AAC ACG 2 34
Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr

70 75

CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACT 273

Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr
80 85 90

GCA GTG, TAT TAC TGT GCA TCT CTC ATC TAC TAT GGT TAC 312
Ala Val Tyr Tyr Cys Ala Ser Leu lie Tyr Tyr Gly Tyr

95 100

GAC GGG TAT GCT ATG GAC TAC TGG GGC CAA GGT ACC CTG 3 51
Asp Gly Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu
105 110 115

GTC ACC GTG AGC TCA 3 66

Val Thr Val Ser Ser

120

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US93/08435

A. CLASSIFICATION OF SUBJECT MATTER
IPC(5) : Please See Extra Sheet.

US CL :435/240.2, 320.1, 240.27; 424/85.8; 530/387.1, 388.6.
According to International Patent Classification (IPC) o r to both national classification and IPC

B. FIELDS SEARCHED ~

Minimum documentation searched (classification system followed by classification symbols)
U.S. : 435/240.2, 320.1, 240.27; 424/85.8; 530/387.1, 388.6.

Documentation searched other than minimum documentation to the extent that such documents are included rn the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
CAS, Bios is, Medline.

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No,

EP, O, 270,077 (Nakatani et. al.) 06 August 1988, see entire
document.

The EMBO Journal, Volume 5, No. 7, issued 1986, Andrew J.
Caton et. al., "Structural and functional implications of a restricted
antibody response to a defined antigenic region on the influenza
virus hemagglutinin", pages 1577-1587, see figures 4 and 5.

6,16-18,25,26

6, 16-18, 25,26

1 x[ Further documenti are listed in the continuation of Box C. | | See patent family annex.

•A*

•L'
•O"

Special catego ri e s of cited

document defining the general state of the ait which is not
to be part of particular relevance

earlier document published on or after the international filing date

document which may throw doubts on priority ckum(s) or which is
cited to establish the publication date of another citation or other
special reason (as specified)

•T

"X-

"Y-

document referring to an oral

use, exhibition or other

document published prior to the international filing date but later than
the priority date claimed

later document published after the mternaaonal filing date or priority
date and not in conflict with the application but cited to understand the
principle or theory underlying the invention

document of particular relevance; the claimed invention cannot be
considered novel or cannot be considered to involve an inventive step
when the document is taken alone

document of particular relevance; the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such combination
being obvious to a person skilled in the art

document member of the same patent family

Date of the actual completion of the international search

16 December 1993

Date of mailing of the international search report

27 DEC -893

Name and mailing address of the ISA/US
Commissioner of Patents and Trademarks
Box PCT

Washington, D.C. 20231
Facsimile No. NOT APPLICABLE

Authorized officer

PAULA HUTZELL

Telephone No. (703) 308-0196

Form PCT/ISA/210 (second sheet)(July 1992)*

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US93/08435

C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category 1

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

The Journal of Immunology, Volume 139, No.7, issued 01
October 1987, Harout Dersimonian et. al, "Relationship of human
variable region heavy chain germ-line genes to genes encoding
anti-DNA autoantibodies", pages 2496-2501, see page 2498.

Bull World Health Organ, 68 Suppl. issued 1990, Mellouk et. al.
"Evaluation of an in vitro assay aimed at measuring protective
antibodies against sporozoites", pages 52-59, see entire abstract.

Proceedings of the National Academy of Sciences, Volume 86,
issued December 1989, Queen et. al. "A humanized antibody that
binds to the interleukin 2 receptor", pages 10029-10033, see
entire document.

1-27

Form PCTYISA/210 (continuation of second sheet)(Ju!y 1992)*

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US93/08435

A. CLASSIFICATION OF SUBJECT MATTER:
IPC (5):

C07H 21/02, 21/04; C12N 15/70, 15/74, 15/79, 5/10; C07K 15/28; A61K 39/395.

Form PCT/ISA/210 (extra sheet)(July 1992}*

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